Home

Applied Biosystems QuantStudio™ 12K Flex Real

1. The QuantStudio 12K Flex Software performs the same analysis of data from an instrument verification runs for 96 well plate Fast 96 well plate 384 well plate or array card blocks After the run the QuantStudio 12K Flex Software 1 Generates a standard curve from the averaged threshold cycle Cy values of the replicate groups of standards Calculates the concentration of the two unknown populations using the standard curve Calculates the following to assess the QuantStudio 12K Flex System performance CopyUnk 3 0CopyUnk2 gt CopyUnk 3 6copyunk1 where e CopyUnk Average copy number of unknown population A OcopyUnk1 Standard deviation of unknown population A e CopyUnk Average copy number of unknown population B 5 OcopyUnk2 Standard deviation of unknown population B Note Unknown population A refers to the 5 000 copy population in columns 7 15 of the TaqMan RNase P Plate or the 800 copy population in rows C and D of the loaded array card Unknown population B refers to the 10 000 copy population in the wells of the TaqMan RNase P Plate or the 1 600 copy population in rows E and F of the loaded array card 64 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Installation specification Preparing the verification consumable Preparing the TaqMan RNase P Instrument Verificat
2. Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 83 Calibrating OpenArray Plate Sample Blocks Background calibration Background calibration Required materials When to perform the calibration About the background calibration data IMPORTANT Perform the following procedure only if you are calibrating a QuantStudio 12K Flex System with an OpenArray plate sample block TM During a background calibration the QuantStudio 12K Flex System e Performs two reads of the QuantStudio 12K Flex OpenArray Calibration Plaque for 10 minutes at 60 C e Averages the spectra recorded during the run and extracts the resulting spectral component to a calibration file The QuantStudio 12K Flex Software then uses the calibration file during subsequent runs to remove background fluorescence from the run data e QuantStudio 12K Flex OpenArray Calibration Plaque e Powder free gloves e Safety goggles Perform the background calibration monthly or as often as necessary depending on instrument use During the background calibration the QuantStudio 12K Flex Software captures a series of images of the black side of the OpenArray Calibration Plaque using each instrument filter The software measures the fluorescence across the image A background calibration passes if the collected images for all filters have signals that are within normal range I
3. 1 While viewing the background calibration data in the Analysis screen select the QC tab and review the list of wells that failed the quality check Rotate the background plate 180 then perform the background calibration again Determine the location of the contaminated wells again If the position s of the contaminated well s in step 1 and step 2 are e Identical The sample block is contaminated Decontaminate the sample block e Reversed The background plate or array card is contaminated Discard the plate or array card then perform the background calibration using a new background plate or array card If the calibration fails after you replace the background plate and decontaminate the sample block a Cover a plate or array card with a piece of black paper b Perform the background run as explained in this chapter substituting the plate or array card covered with paper for the background plate or array card c After the run is complete and while viewing the calibration data select all wells in the Plate Layout tab then view the Spectral plot for the peak s If the peak associated with the contamination is e Visible The optics of your QuantStudio 12K Flex System may be contaminated Contact Life Technologies for further support e Absent The sample block is contaminated Decontaminate the sample block again and repeat the calibration 80 Applied Biosystems QuantStudio 12K Flex Real Time PC
4. IMPORTANT The application of the syringe tip requires force Confirm that the tip is locked firmly in place before proceeding Carefully draw approximately 2 mL of OpenArray FAM Dye Solution into the syringe Grasp the OpenArray calibration case in position 1 by the edges then remove it from the OpenArray Calibration Carrier Remove the RUN EMPTY FIRST label that covers the fill port of the OpenArray calibration case 92 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 3 Calibrating OpenArray Plate Sample Blocks Dye calibration 5 While holding the OpenArray calibration case vertically insert the syringe tip into the fill port at end of the case then dispense the fluid completely in one gentle continuous motion Empty OpenArray calibration case a FILE label indicates the location of the fill port Fill port Note Try to minimize creating air bubbles when you dispense the fluid You can leave one small air bubble at the fill port to prevent overfilling 6 Seal the loading port by inserting an OpenArray Plug into the port and twisting it clockwise until hand tight then remove the handle from the plug A OpenArray plug insert and twist 7 Load the sealed OpenArray calibration case into the same position on the OpenArray Plate Carrier that it previously occupied position 1 IMPORTANT You must load the f
5. 1 Plug the USB drive that contains the instrument settings into the USB port on the right side of the QuantStudio 12K Flex Instrument touchscreen 2 If the instrument is in standby touch the QuantStudio 12K Flex Instrument touchscreen to activate it then press 4 3 In the Main Menu touch Tools then touch a Restore Settings 164 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix A Manual Instrument Operation Maintaining the instrument from the touchscreen 4 In the Restore Settings screen select the settings to restore a Touch the settings that you want to restore from the list b Touch Restore to upload the instrument settings from the USB drive IMPORTANT Do not remove the USB drive from the QuantStudio 12K Flex Instrument until you are instructed to do so Note Alternatively touch Restore Default Settings to restore the QuantStudio 12K Flex Instrument to the factory settings 5 After the QuantStudio 12K Flex Instrument reboots unplug the USB drive Performing an You can use the QuantStudio 12K Flex Instrument touchscreen to perform a instrument self comprehensive self test of the QuantStudio 12K Flex Instrument subsystems After test the self test is complete the QuantStudio 12K Flex Instrument generates two files that provide a detailed summary of the instrument condition and function In the event of a probl
6. Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 263 Glossary reporter reverse primer reverse transcriptase Rh ROX dye rs number run method sample sample definition file Sample Library sample SNP assay reaction sample target reaction 264 A fluorescent dye used to detect amplification With TaqMan reagents the reporter dye is attached to the 5 end With SYBR Green reagents the reporter dye is SYBR Green dye SYBR and HRM specific dyes are DNA binding dyes An oligonucleotide that flanks the 3 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target An enzyme that converts RNA to cDNA See normalized reporter Rn A dye supplied by Life Technologies and precalibrated on the instrument ROX dye is used as the passive reference See refSNP ID Definition of the reaction volume and the thermal profile for the instrument run The run method specifies the temperature time ramp and data collection points for all steps and stages of the instrument run The biological tissue or specimen that you are testing for a target gene A tab delimited file txt or csv that contains the following setup information well number sample name and custom sample properties In the software an editable collection of sample names to use in experiments The samples in th
7. If an event triggers notification the next time any user with an Administrator role logs in the software lists those events indicating the time each event occurred and the user who triggered the event The Administrator has the option of acknowledging the event which removes it from the notification list s Email Notification If an event triggers notification the QuantStudio 12K Flex Software sends an email to the addresses in the adjoining Email Address column of the table The email notification displays the triggered event and displays the time that the event occurred and the user who triggered the event 5 Click OK Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 141 6 Security Audit and Electronic Signature Managing user accounts Managing user accounts Creating and The software includes a default Administrator user account with permissions defined editing user by the account user role to perform all functions in the software You cannot modify accounts this account Action Procedure Create a user 1 In the QuantStudio 12K Flex Software select Tools gt Security gt account Settings 2 In the Security Settings dialog box select the Users tab 3 Click Create to display the New User dialog box 4 Enter user name password first name middle initial optional and last name Click a field to display the field limits which are specified
8. Indicates the presence of moving parts and to proceed with appropriate caution Indicates the presence of a biological hazard and to proceed with appropriate caution Indicates the presence of a laser light in the instrument and to proceed with appropriate caution Indicates the presence of an ultraviolet light and to proceed with appropriate caution Indicates the presence of a slipping hazard and to proceed with appropriate caution Indicates the presence of a radiological hazard and to proceed with appropriate caution gt PPP EEL i Environmental symbols on instruments The following symbol applies to all Life Technologies electrical and electronic products placed on the European market after August 13 2005 Symbol Description P Do not dispose of this product as unsorted municipal waste Follow local f municipal waste ordinances for proper disposal provisions to reduce the l environmental impact of waste electrical and electronic equipment WEEE _o6 European Union customers Call your local Life Technologies Customer Service office for equipment pick Hsp and recycling See www lifetechnologies com for a list of customer service offices in the European Union Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 239 C Safety Instrumentation safety Locations of safety The QuantStudio 12K Flex Instrument contains warning
9. must be renewed regularly If a license has expired or is nearing expiry the TM QuantStudio 12K Flex Software displays a warning when the software is started Note License keys are found on the QuantStudio 12K Flex Software CD packaging or they can be supplied by Life Technologies support Monitoring the current licenses You can use the QuantStudio 12K Flex Software to review the status and expiration date of the licenses currently installed to the software 1 In the main menu of the QuantStudio 12K Flex Software select Tools License Central 2 In the License Central dialog box review the status of your licenses The software displays the status of all installed licenses where possible states include Current and Expired and the date at which it expires Note The License Central dialog box lists the QuantStudio 12K Flex Software core application and modules on different rows because the licenses are maintained separately 3 Optional Save the license information to a text file a Select the license that you want to export from the table then click Save License Request Info b Navigate to the appropriate location then click Save 4 When you are done click OK Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 117 118 Maintenance Managing QuantStudio 12K Flex Software licenses Renewing a license If you have a valid license k
10. Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Troubleshooting Troubleshooting S Table 2 Troubleshooting ROI calibrations 066 eens 72 S Table 3 Troubleshooting background calibrations 08 73 S Table 4 Troubleshooting uniformity calibrations s 0 cea 74 m Table 5 Troubleshooting dye calibrations 0666s 75 S Table 6 Troubleshooting normalization calibrations e ssssuao 76 m Table 7 Troubleshooting RNase P instrument verification experiments 77 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 71 CL olpnjgjueng SU 91515019 panddy WL aping J SN uonesjsiuipy pUe aueuajulew Waj skS YJd awil j eay X84 NZ1 Table 2 Troubleshooting ROI calibrations Problem symptom Possible cause Action ROI calibration failed ROI image is faint The sample block or heated cover may not be seated correctly 1 2 3 Power off and unplug the QuantStudio 12K Flex Instrument Wait for 15 minutes then open the access door Firmly push the sample block and the heated cover toward the back of the QuantStudio 12K Flex Instrument to confirm that they are seated correctly IMPORTANT Confirm that the arrows on the front handle of the heated cover align as shown below If the arrows do not align push the heated cover further into the QuantStudio 12K Flex Instrument until the hand
11. Correct Incorrect Za Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 185 Calibration Consumable Preparation Creating a custom dye plate for calibration Adding the custom 1 Start the dye calibration dye to the software a In the Home screen of the QuantStudio 12K Flex Software click Instrument Console b In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments c Select the QuantStudio 12K Flex Instrument then click Manage Instrument d In the Instrument Manager click Maintenance then click Dye e In the Background Calibration screen click Start Calibration 2 In the Dye window select a custom dye from the list or create the custom dye a Click New Dye b In the Dye Library dialog box click New c Complete the New Dye dialog box then click OK Field option Action Name Enter a name for the custom dye Wavelength Enter the wavelength at which the dye fluoresces Type Select s Reporter if the dye works in conjunction with a quencher dye to report an increase of PCR product e Quencher if the dye suppresses the fluorescence of a reporter dye until amplification of PCR product Both if the dye reports an increase of PCR product wit
12. The convention can consist of all or some of the following interchangeable arguments in any order Custom Name Field The alphanumeric string specified by the c parameter ID The barcode of the plate specified in the barcode file specified by the b parameter Example f Custom Name Field_ID If the parameter is used without arguments then the software names files according to the following convention Custom Name Field_ID lt dirpath gt Required Specifies the path of the directory lt dirpath gt to which the software saves the new files Example 1 C Applied Biosystems QuantStudio 12K Flex Software User Files experiments 194 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix D Command line Software Operation D Command syntax and arguments Before creating experiment files the software confirms whether the export location exists and aborts if the location does not exist m lt filepath gt Optional Specifies the path and name lt filepath gt of the sample file that the software uses to create new files Example m C samplefiles samplefile txt n lt integer gt Optional If the b parameter is not included specifies number of experiments lt integer gt that the software will create If no value is supplied the software creates 25 experiments by default Example n 31 s lt filepath gt Op
13. concentration a In the center of a 96 384 well plate prepare a dilution series of the custom dye for example 25 50 100 200 400 800 1600 and 3200 nM using 20 uL volumes for a 96 384 well plate b Seal the reaction plate using an optical adhesive cover c Load the prepared reaction plate 2 Start the calibration wizard a In the Home screen of the QuantStudio 12K Flex Software click Instrument Console b In the Instrument Console select your QuantStudio 12K Flex Instrument then click Add to My Instruments c Select the QuantStudio 12K Flex Instrument then click Manage Instrument d In the Instrument Manager click Maintenance then click ROI e In the ROI Calibration screen click Start Calibration f In the ROI dialog box click Next until prompted to load the QuantStudio 12K Flex Instrument When the side door opens load the sealed plate Ensure that the plate array card is properly aligned in the holder 184 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix C Calibration Consumable Preparation Creating a custom dye plate for calibration g In the ROI dialog box select Check the box when the ROI calibration plate has been loaded click Next twice then click START RUN to start the calibration 3 When the run is complete inspect the ROI images a Select the first filter from the Filter drop down list b Record th
14. eouezejoy eaTssed x odAI quewnazq4sur x sa dwexy 12K Flex Real Time PCR System Maintenance and Administration User Guide TM Applied Biosystems QuantStudio 204 E Appendix E File Format Reference Plate setup file format aOW OAN aOW OAN aOW OAN aOW OAN aOW OAN aOW OAN aOW OAN a OW OAN a OW OAN a OW OAN ZETSTTW 19 10d ZETSTTW FOTOD ZETSTTV SWEN ZETSTTY TeyoueNnd TeTeTTw 19 7170d TeTSeTTW AOTOD TeTeTIW SWeN TeTSTIV MY 1 0 0S 0 d9uu Y a9W IN 0706701999 Y aOW OAN 0706701999 Y aOW OAN 0706701999 Y aOW OAN 0 0S 0 98u Y aOW OAN 0 0S 0 98u Y aOW O4AN 0 0S 0 98u Y aOW O4AN 0 0S 0 98u Y aOW O AN 0 0S 0 49 uu Y aOW O AN 0 0S 0 98u Y aOW O AN OIA OIA OIA OIA OIA OIA OIA OIA OIA OIA u 0S 0 0 9uu u 0S 0 0 999 u 0S 0 0 999 u 0S 0 0 999 u 0S 0 0 999 u 0S 0 0 999 u 0S 0 0 999 u 0S 0 0 duu u 0S 0 701999 u 05 040 999 NMONYNO Z TITI Od Z TITI Od Z AISTIV Od T TITI Od T S1aTIW Od T TITI od DIN DIN DIN LOR R UR u 0 SL 0 98u veestsa 07 6L 701999 PEGSTSI 07 6L 701999 PEGSTSI 07 6L 701999 PEGSTSI 0 SL 0 duu pe6stsa 0 SL 0 duu peestsa 0 SL 0 duu pe6stsa 0 SL 0 duu EGSTSI 0 SL 0 998a EGSTSI 0 SL 0 998a EGSTSI yseL roToD Aessw aNs owen Aessy dNS dNs dNs dNs dNS aNS aNS daNS daNS daNS daNS sajdurexy DIN OAN Jo
15. 0 S7 0 d5uu ToruUCD 0 S7 0 d5uu TorUCD 0 SZ7 0 d5uu ToruUCD 0767 01999 TOI3 U09 0767 01999 TOI3 U09 0707671999 Jor 0 0 SZ 999 qTory 0 0 SZ 999 qory 0 0 SZ 999 qTory 0 0 SZ 999 TOI 0 0 SZ 999 TOI sog sod sod sod sod sod USR uop uoo uoo uop uop owen eTdues ANANNNTTMNNWOYO T TTSM dnjes at dues XOA eouezejoy AFSSLd x XOTANZLOTpNyAsquend edAy queumazqsul x sajdurexy dU oHUOD atsod yeusayur ue pue aduenbes drw0uas onbrun e Mde 01 saqgoid gue bey payeqe oAp DIA PUL p NWA sasn Aesse uoreyep L ZH ZSTO 10 41 uesouyed e jo aouasaid ay 101 s jdwres suaaios zu wr dx ay W S S X ZI ne OLPMaS END L UO UNL aq 0 JUBUTTIadxa adUasqe aduUasaid e 107 paean apy das ejd e smoys jdwex SULMOTIO aU s u Wj dx aduasqe aduasaid 205 12K Flex Real Time PCR System Maintenance and Administration User Guide TM Applied Biosystems QuantStudio E File Format Reference Sample file format Sample file format File structure Example file The QuantStudio 12K Flex Software can import sample files to populate sample information into an open experiment A sample file is a tab delimited ASCII text file txt that contains sample well designations and custom sample properties The files can be created manually using a text processor or generated automatically by third party applications IMPORTANT To guarantee successful import the file
16. 0 1 mL 125 strips 4358293 MicroAmp Fast Optical 96 Well Reaction 10 plates 4346906 Plate with Bar Code 0 1 mL 200 plates 1366932 MicroAmp Optical 96 Well Reaction Plate 10 plates N8010560 oem 500 plates 4316813 MicroAmp Optical 96 Well Reaction Plate 10 plates 4306737 with Bar Code 0 2 mL 500 plates 1326659 MicroAmp Optical 384 Well Reaction Plate 1000 plates 4343370 MicroAmp Optical 384 Well Reaction Plate 1000 plates 4343814 withBar Gode 500 plates 4326270 50 plates 4309849 MicroAmp Optical 8 Cap Strip 300 strips 4323032 MicroAmp Optical 8 Tube Strip 0 2 mL 1000 tubes 4316567 MicroAmp Optical Adhesive Film 4311971 236 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Safety This appendix covers Instrumentation safety 0 6 6 eee eee eens 238 Symbols on instruments 2 apele atin EEEE eee eens 238 Locations of safety labels on instruments 00 00 eee eee eee ee eee 240 General instrument safety 2 0 NR TRS R X R E R irr einki anir TR 241 Physical hazard safety TTT 242 Electrical safety v 20 ccs aid prad haa iiaia ole da iia ai a aai dare 242 Bar code scanner laser safety n u nunnan nnr eee ees 243 Workstation Safety semer pirane tti upian ua EENES EEE RETENER NEE EENE N 243 Safety and electromagnetic compatibility EMC standards 244 Chemical safety 6 ccc nnn nee EE ee 245 General chemical Saf
17. 0701999 0 S2 86 duu 01 0 0 999 0 S2 86 duu u GOT 0701999 7677961999 SUT 0701999 vL0 5Z 9619909 ZOTOD 4 612L THD UWAOO THD UWA THD UvA THD UvA THD AVAD TH UWAOO wey Jebrel ZoToD dnozrbotg u 0 SZ 0 9uu u 0 SZ 0 9uu 0767 01999 0767 01999 u 0 SZ 0 98u u 0 GZ 01 999 u 00 SZ 999 u 00 SZ 999 u 0 0 SZ 999 u 0 0 SZ 999 u 0 0 SZ 999 u 0 0 GZ 98u eue dnozbotg ZoToD etTdues xou sajduexy QOW OAN Joypuanb yuadsasonyj uou ay pue adp Jajz10dar NVA 9Y YM paiad gi are saqoid y shesse gueypybey yyoq 104 Tom owes ay 0 poppe are S199161 HD pue YYNOD 831V OM ay 101 sAesse ay ITM aAoge JuauLIadxa ayy Jo UOTSIaA x jdymu e 101 apy dnjas ayeyd e smoyus afdurexa SuLMoT OJ au qOW O AN aOW O AN aOW OAN aOW OAN aOW O AN aOW OAN aOW O 4N 9W AN 9W AN aOW OAN aOW OAN aOW OAN a OW OAN 9W AN squewuog AqtTquend zAseyouend File Format Reference Plate setup file format WW WW zeqroday NMONANNA NMONANN NMONANNA NMONANN NMONANN NMONANN NMONANN SNONASOGNa SNONASOGNa SNONASOGNG SNONASOGNG SQONASOCNG SNONASOGNa SNONASOGNaA KI u SOT 0 0 u SOT 070 909 u Od SOT 0701999 SOT 0701999 u GOT 0 0 98u SUL 0701999 ua SOT 0701 999 07677861 999 0 SZ 86 duu u 0 SZ 86 G94 0767961909 0 SZ 8
18. 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Preparing array cards for instrument calibration Preparing array cards for instrument calibration IMPORTANT Perform the following procedure only if you are verifying the performance of a QuantStudio 12K Flex System with an array card sample block Required materials e QuantStudio 12K Flex System Array Card Spectral Calibration Dye Kit Array Cards empty Array Card Spectral Calibration Dye Kit including FAM dye mix VIC dye mix ROX dye mix ROI dye mix Background Buffer FAM ROX dye mix and VIC ROX dye mix e Applied Biosystems Array Card Staker Sealer e Centrifuge with array card buckets and array card carrier clips e Permanent marker or pen e Pipettor 200 uL with pipette tips e Powder free gloves e Safety glasses Filling the IMPORTANT Wear powder free gloves while creating the calibration array cards calibration array cards Note This procedure explains how to create all of the array cards required to calibrate the QuantStudio 12K Flex System but not all of them are required for a monthly maintenance Before preparing array cards for calibration see Recommended calibration and maintenance on page 36 to determine which calibrations are required Note You can view a video of the array card loading procedure on the Life Technologi
19. 12K Flex System or computer It does not protect against a power surge or failure Uninterruptible power supply UPS We recommend the use of a 1 5 kVA uninterruptible power supply UPS especially in areas prone to power failure Power failures and other events that abruptly terminate the function of the QuantStudio 12K Flex System can corrupt data and possibly damage the computer or the instrument IMPORTANT UPSs provide power for a limited time They are meant to delay the effects of a power outage not to serve as replacement power sources In the event of a power loss power off the instrument and the computer unless you expect to regain power within the battery life of the UPS Surge protector We recommend the use of a 10 kVA surge protector line conditioner in areas with frequent electrical storms or near devices that are electrically noisy such as refrigerators air conditioners or centrifuges Short duration high voltage power fluctuations can abruptly terminate the function of and thereby damage the components of the computer and the QuantStudio 12K Flex Instrument Note A dedicated line and ground between the QuantStudio 12K Flex System computer and the building s main electrical service can also prevent problems caused by power fluctuations Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 29 1 Getting Started QuantStudio 12K Flex Sys
20. Cleaning the WARNING PHYSICAL INJURY HAZARD During instrument operation the sample block sample block and heated cover can be heated to 100 C Before removing the sample block be sure to wait until it reaches room temperature IMPORTANT Wear powder free gloves when you perform this procedure 1 Identify the contaminated wells of the sample block see Identifying contamination on page 80 2 Power off and unplug the QuantStudio 12K Flex Instrument then allow it to cool for 15 minutes 3 Open the access door 4 Firmly press down on the handle of the sample block then remove it from the QuantStudio 12K Flex Instrument Place the sample block on a clean dry surface 5 Clean the contaminated wells of the sample block using deionized water a Pipet a small volume of deionized water into each contaminated well b In each well pipet the water up and down several times to rinse the well c Pipet the water to a waste beaker d Using a cotton swab scrub inside of each contaminated well If you are decontaminating an array card or OpenArray plate sample block swab the surface of the block that contacts the consumable e Using a lint free cloth absorb the excess deionized water 112 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 6 10 11 12 13 14 Chapter 4 Maintenance 4 Decontaminating the sample block Load
21. Click Next 2 Enter any comments you have in the Comments field click Finish then click Yes when prompted to save the results Passed Failed 1 Repeat the calibration If necessary clean the OpenArray Calibration Plaque before you repeat the calibration as described in Caring for the OpenArray Calibration Plaque on page 83 2 If the calibration fails again contact Life Technologies for further assistance When the instrument door opens remove the OpenArray Calibration Plaque from the instrument tray H WARNING PHYSICAL INJURY HAZARD During instrument operation the plates or plaque can reach 100 C Ensure the plate or plaque is at room temperature before removing Return the OpenArray Calibration Plaque to its original packaging or a clean plastic bag IMPORTANT Do not expose the OpenArray Calibration Plaque to sunlight for extended periods of time When not in use store the plaque at room temperature in the original packaging in a clean dark location IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the OpenArray Calibration Plaque remove the plate as explained in Troubleshooting on page 105 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 89 Calibrating OpenArray Plate Sample Blocks Dye calibration Dye calibration Required materials When to perform the dye calibrations IM
22. Contact Life Technologies to order a replacement TaqMan RNase P plate or array IDSL MAES stopped card kit Repeat the experiment and make sure to properly seal and centrifuge the functioning RNase P plate If the replacement RNase P plate or array card fails contact Life Well displays the NOISE flag e Droplets on the sides of the Technologies for further assistance indicating that the well produced wells more noise in the amplification e Improper sealing or seal leaks plot than other wells on the plate e Condensation on the reaction plate Well displays the OUTLIERRG e Contamination 1 Decontaminate the work area and pipettors flag indicating that the C ofthe e Improper sealing or seal leaks 2 Contact Life Technologies to order a replacement TaqMan RNase P plate or array well deviates significantly from C values in the associated replicate group only the outlier is flagged card kit Repeat the experiment and make sure to properly seal the RNase P plate or array card If the replacement RNase P plate or array card fails contact Life Technologies for further assistance Well displays the SPIKE flag indicating that the amplification curve contains one or more data points inconsistent with the other points in the curve e Bubbles in the reaction e Evaporation during the denaturation step because of improper sealing or seal leaks Contact Life Technologies to order a replacement TaqMan RNase P plate or array c
23. Location For indoor use only IMPORTANT Do not place the QuantStudio 12K Flex Instrument next to electrically noisy devices such as a refrigeration unit or vibration sources such as a centrifuge pump or compressor Excessive vibration can affect instrument performance T The QuantStudio 12K Flex Instrument can be used in an environment that contains nonconductive pollutants only dust particles or wood chips Typical environments with a Pollution Degree II rating are laboratories sales and commercial areas Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide QuantStudio 12K Flex System layout and connections Chapter 1 Getting Started Specifications and layout The QuantStudio 12K Flex System consists of the components shown in the following figure Component Description QuantStudio 12K Flex Instrument Performs fluorescence research detection and data collection of experiment and calibration consumables Computer L Monitor Keyboard Mouse Run the QuantStudio 12K Flex Software that is used to e Calibrate the QuantStudio 12K Flex Instrument e Set up experiments e Optional Run experiments e Analyze experiments Barcode reader Scans the barcodes of consumables before they are loaded into the QuantStudio 12K Flex Instr
24. Program Files Applied Biosystems QuantStudio12KFlex docs double click README html then read the QuantStudio 12K Flex Software Release Notes for the compatibility of interest 2 Ifthe release notes do not mention the compatibility visit www lifetechnologies com quantstudio then search the website for the compatibility of interest 3 If the website does not contain the information of interest contact Life Technologies Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 115 4 Maintenance Updating the QuantStudio 12K Flex Software and Firmware Updating the QuantStudio 12K Flex Software and Firmware Updating the QuantStudio 12K Flex Software Updating the QuantStudio 12K Flex Instrument firmware TM Life Technologies may release updates to the QuantStudio 12K Flex Software and QuantStudio 12K Flex Instrument firmware that you can install without the aid of Life Technologies service personnel You can obtain updates directly from the service section of the Life Technologies website For the latest services and support information for the QuantStudio 12K Flex System 1 Go to www lifetechnologies com support software 2 In the Software Downloads page select Applied Biosystems QuantStudio 12K Flex Real Time PCR System from the menu 3 In the QuantStudio 12K Flex Instrument Software Downloads page click Updates Patches The website ope
25. Security gt Settings In the Security Settings dialog box select the Audit tab Select or deselect Enable Audit Pe a o Click Apply Settings Selecting objects to 1 Select the objects to audit and the mode for each enabled item audit e Experiments e Experiment Templates 2 Set the Audit Mode for each item you enable for auditing e Optional The event is audited a reason prompt is displayed but the user can cancel and continue without entering a reason e Required The event is audited a reason prompt is displayed and the user must specify a reason e Silent The event is audited no reason prompt is displayed 3 Click Apply Settings Creating audit You can create modify and delete the reasons that are available for selection in the reason settings Audit Reason dialog box displayed when a user performs an audited action 1 To require users to select a pre defined reason in the Audit Reason dialog box displayed when a user performs an audited action enable Require users to select a reason to change from the list Users are not permitted to enter a reason 2 As needed click Create or select a reason from the list then click Edit or Delete Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 145 6 Security Audit and Electronic Signature Generating audit reports Generating audit reports Displaying audit histories from the Security Settings
26. and check each corresponding through hole in the reaction plate for evaporation or low reaction volume Order and perform a replacement RNase P experiment If the replacement RNase P OpenArray plate fails contact Life Technologies for further assistance Through hole displays the SPIKE flag the amplification curve contains one or more data points inconsistent with the other points in the curve e Bubbles in the reaction e Evaporation during the denaturation step because of improper sealing or seal leaks Order and perform a replacement RNase P experiment Properly seal and centrifuge the RNase P OpenArray plate If the replacement RNase P OpenArray plate fails contact Life Technologies for further assistance s 20 g ajduwes a ejq Aeiuyuedg buneiqneg saydeyg 6unooysayqnos E 901 olpnysjueng swas soig panddy WL aping Jas uoNesSiuiWpy DUS aueuajuleW WaISkS Yd awi jeay X9 4 YZ Problem symptom Possible cause Action Through hole displays the OFFSCALE flag the fluorescence signal for one or more dyes in the through hole exceeds the instrument s maximum detectable range for one or more cycles e Fluorescent contaminant on the reaction plate or sample block e Fluorescent contaminant in the reaction Perform a background calibration If you detect fluorescent contamination decontaminate the sample block Order and perform a replacement RNase P experiment If the r
27. andar o template Unknown A Unknown B STD controls NTC 96 well platet 6 6 1 0 384 well plate 10 10 2 0 Array card 4 4 4 0 5 000 copy population for 96 384 well plates 800 copy population for array cards B 10 000 copy population for 96 384 well plates 1 600 copy population for array cards Maximum number of wells that can be removed from each standard population Standard 96 well plates or Fast 96 well plates d Repeat step 3a through 3c for each replicate population unknowns standards and no template controls on the plate or array card Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 69 Calibrating Multi Well Plate and Array Card Sample Blocks Verifying the instrument performance 9 Review the Results Table for quality flags generated by the experiment a Select the Results Table tab b Review the Flag column for wells that generated quality flags c Troubleshoot each well that generated a flag as explained in Table 7 Troubleshooting RNase P instrument verification experiments on page 77 e AMPNC Amplification in negative control e BADROxX Bad passive reference signal e BLFAIL Baseline algorithm failed e CTFAIL Cy algorithm failed e EXPFAIL Exponential algorithm failed e HIGHSD High standard deviation in replicate group e NOAMP No amplification e NOISE Noise higher than others in plate e NOSIGNAL No signal i
28. contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks e Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse e Position the keyboard mouse and monitor to promote relaxed body and head postures Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 243 C Safety Instrumentation safety Safety and This section provides information on electromagnetic co e U S and Canadian safety standards mpatibility EMC e Canadian EMC standard standards e European safety and EMC standards e Australia and New Zealand EMC standards T U S and Canadian safety standards C s L US The instrument has been tested to and complies with standard UL 61010 1 2nd Edition CSA C22 2 No 61010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements UL 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials Canadian EMC standard This instrument has been tested to and complies with standard ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators Cet appareil numerique de la classe B est conforme a la norme NMB 001 du Canada European safety and EMC standards This instrument meets European requir
29. dialog box You can use the QuantStudio 12K Flex Software to generate reports of audit history from both the Security Settings dialog box and open experiments templates or studies m Displaying audit histories from the Security Settings dialog box 146 S Displaying audit histories for an experiment or template 149 Displaying audit histories 1 2 In the QuantStudio 12K Flex Software select Tools Security gt Settings In the Security Settings dialog box select the Audit tab then click View Reports Note To access the Audit Reports screen the user role for an account must specify the Configure SAE permission Users without the Configure SAE permission can view object audit histories for individual entries in the libraries by selecting entries then clicking View Audit History Select a tab to display e System Configuration History Security audit and electronic signature configuration records including audit history for each user account e Action Record System specified audit events Optional Select Filter by then filter the table e Sort the table e Specify filters date range user name action object or record type object or record name reason then click Refresh Note The Reason field in System Configuration History is not used e Select one or more records then click View Report 146 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Mai
30. if not avoided ZN may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in a Life Technologies document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to Life Technologies instruments see Safety symbols on page 238 The SDSs for any chemicals supplied by Life Technologies or Ambion are available to you free 24 hours a day For instructions on obtaining SDSs see SDSs on page 246 IMPORTANT For the SDSs of chemicals not distributed by Life Technologies or Ambion contact the chemical manufacturer 14 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Safety labels on instruments preceding section About This Guide Safety information The following CAUTION WARNING and DANGER statements may be displayed on Life Technologies instruments in combination with the safety symbols described in the Hazard symbol English Francais A CAUTION Hazardous chemicals Read th
31. in Security Settings Note First name MI middle initial and last name are used to create User Full Name which is displayed as the name of the logged in user Note You cannot change the user name after you save the user account 5 Select Password Expires at First Login to require the user account to specify a new password at first log in The Password Expires On date is specified in Security Settings 6 Select the user role and the electronic signature state determines if a user account has permission to electronically sign objects Leave the status set to ACTIVE 7 Optional Enter email for information only phone and comments 8 Click Save A grayed Save button indicates an invalid entry in a field Click a field to display the limits for the field then enter a valid entry Edit a user 1 In the Users screen select a user account then click Edit accoun Note If you select multiple users only Status and Role will be changed 2 Edit settings as needed You cannot edit the user name of an existing user 3 Click Save Activate a 1 In the Users screen select the user suspended user 2 Click Edit account 3 Change the Status from SUSPENDED to ACTIVE then click Save Disable IMPORTANT You cannot delete a user because user records are inactivate a required for auditing To disable a user account inactivate it as follows user account 1 In the Users screen select the user 2 Click Edit 3
32. when security is disabled 150 electronic signature users is signed field 155 signing 155 EMC standards 244 enable calibration reminders 134 electronic signature 150 security instrument 172 security software 139 endogenous control 257 endpoint read 257 ergonomics safety 243 error 257 e sig See electronic signature Ethernet port 25 126 129 define IP settings 170 Index experiment document 257 name 257 type 18 257 experiments archive 110 create from touchscreen 159 RNase P instrument verification 63 95 run from touchscreen 160 transfer to from the instrument 133 161 export 257 7900 file format 223 audit records 149 audit settings 153 e sig settings 153 QuantStudio file format 209 RDML file format 228 security settings 153 user account settings 153 export formats 208 7900 file 223 QuantStudio file 209 RDML file 228 F FAM dye 19 53 91 fans instrument 25 feet 24 27 file assay information 191 207 barcode 191 207 export formats 208 import formats 200 plate setup 201 sample 191 206 setup 191 fill array cards calibration 37 instrument verification 37 66 filter 258 filter sets 19 firmware update 116 166 flag 258 fluorescence background 45 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 271 Index format 7900 export file 223 assay information file 207 barcode file 207 plate setup file 201 QuantStudio export file 209 RDML export f
33. 127 overview 126 settings instrument 170 no amplification control NAC 260 no template control 260 nonfluorescent quencher minor groove binder 260 normalization calibration 59 260 data 59 perform 60 troubleshoot 76 when to perform 59 normalized quantity 260 normalized quantity mean 260 normalized quantity SE 260 normalized reporter Rn 260 notifications maintenance reminders 169 security auditing electronic signature 140 NTC 260 0 object audit history display 146 149 152 omit outliers 68 omit well 260 online Help See Help system OpenArray AccuFill System initialize 97 OpenArray Calibration Cases guidelines for handling 91 OpenArray plate load 99 loading 98 99 serial number 98 OpenArray Plate RNase P Kit 95 operating system update 115 optical calibration perform 51 troubleshoot 73 order calibration and verification kits 233 from the software 231 from the website 231 how to 230 outlier 260 removal 68 removal for installation specification 65 overvoltage category rating 242 P passive reference 261 password administrator 138 changing 154 expiration 139 restrictions 139 pdf action log 146 149 152 audit reports 148 perform background calibration 47 dye calibration 56 91 normalization calibration 60 optical calibration 51 RNase P instrument verification 68 103 ROI calibration 43 uniformity calibration 51 permissions user account 143 154 physical hazard safety 242 plaque ca
34. 12K Flex Software can export setup and results data from experiment files eds in several file formats that allow further downstream analysis The export formats feature standardized data structures and markup to maximize accessibility by downstream applications The QuantStudio 12K Flex Software supports the following export formats File format Description See QuantStudio12KFlex A QuantStudio12KFlex formatted text file that contains page 209 export file setup and or results data exported from an experiment file eds 7900 export file A legacy 7900 formatted text file that contains setup page 223 and or results data exported from an experiment file eds RDML export file A compressed XML file that contains setup and or page 228 results data exported from an experiment file eds and parsed in Real time PCR Data Markup Language RDML The file is stored as a compressed file using the PKZIP archive format Export formats and the QuantStudio 12K Flex Software API The export formats can be used in combination with the QuantStudio 12K Flex Software application programming interface API to integrate the QuantStudio 12K Flex System into a laboratory information management system LIMS workflow 208 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix E File Format Reference E QuantStudio12KFlex export format QuantStudio12KFlex exp
35. 96 384 Sample Name The name of the sample contained by the well 100 character string SNP Assay The name of the SNP assay added to the well 100 character string Name Task The task assigned to the target in the well UNKNOWN or NTC Allele1 Rn The raw fluorescence associated with the allele 1 Float probe of the SNP assay at the well normalized to the fluorescence of the passive reference dye Allele2 Rn The raw fluorescence associated with the allele 2 Float probe of the SNP assay at the well normalized to the fluorescence of the passive reference dye Pass Ref The raw fluorescence of the passive reference at Float the well Quality The confidence of the automatic allele call Float 1 100 Call The allele call assigned to the sample at the Homozygous specified well lt allele x allele x gt Heterozygous lt allele x allele y gt or Negative Control NC Method The method used to call alleles Auto Or Manual Allele1 Whether the allele 1 threshold was determined true or false Automatic Ct automatically true or manually false Threshold Allele1 The start cycle used to calculate the baseline Float Baseline Start section of allele 1 Allele1 The end cycle used to calculate the baseline Float Baseline End section of allele 1 Allele2 Whether the allele 2 threshold was determined true or false Automatic Ct automatically true or manually false Threshold Allele2 The firs
36. An important quality value the R value indicates the closeness of fit between the standard curve regression line and the individual Cy data points from the standard reactions A value of 1 00 indicates a perfect fit between the regression line and the data points The step at which the temperature changes during the instrument run The ramp rate is defined as C per second In the graphical view of the thermal profile the ramp rate is indicated by a diagonal line Speed at which the temperature ramp occurs during the instrument run Available ramp speeds include fast and standard A plot of raw fluorescent signal as detected through each emission filter used to view raw data for individual wells and at individual cycles A solution that contains all components to run the PCR reaction except for the template sample standard or control Also called a PCR cocktail The PCR reaction components used to amplify the target and to detect amplification Process of collecting fluorescence data during PCR Data from the real time PCR are used to calculate results for quantification experiments or to troubleshoot results for genotyping or presence absence experiments A reporting format that is compliant with the Minimum Information for Publication for Quantitative Real Time Experiments MIQE guidelines In an HRM experiment the melt curve selected by a user in the difference plot to use as a basis for comparison The software displ
37. Baseline automatically true or manually false Baseline Start The first cycle used to calculate the baseline Integer Baseline End The last cycle used to calculate the baseline Integer Custom 1 Custom6 The contents of the custom text fields found in the Results table of the experiment 1024 character string per field If analysis flags are present results data is present in additional columns named for the associated flags true Or false Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix E File Format Reference E QuantStudio12KFlex export format Biological replicate results The following table describes the results data exported from high resolution melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Biogroup The name of the biological replicate group 100 character string Name Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN Or NTC RQ The relative quantity calculated for the replicate Float wells of the target sample combination RQ Min The minimum relative quantity calculated for the Float
38. Click Export 3 Specify aname and location for the export txt file then click Save Note If you export audit records for samples that are not in their original location samples have been deleted or moved an error message is displayed Return sample data files to their original location then export again Displaying audit Displaying the audit history histories for an experiment or template 2 In the open experiment or template click ve Audit then click Audit Records 1 Open an experiment eds or template edt file 3 Optional Filter the table To view fewer records a Check the Filter by check box b Enter criteria for the records of interest such as a date range a user name or a type of action c Click Refresh To view details for a specific record a Click the row in the list on the left to view the details of the record in the table on the upper right b Click any row to view details for individual records in the table on the bottom right Exporting audit records 1 Open an experiment eds or template edt file 2 In the open experiment or template click o Audit 3 In the table on the left select the records to be exported e Click in the table then press Ctrl A to select all the records in the table e Click and drag or press Shift to select continuous rows e Press Ctrl to select discontinuous rows 4 Export the records e Click Export Summary to export only the records in the l
39. Configuration Log into user sessions that have timed out No No Yes Perform E Signing Yes Yes Yes Editing a user role 1 In the Roles screen select a user role then click Edit 2 Edit settings as needed You cannot edit the Administrator user role 3 Click Save Role Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 6 Security Audit and Electronic Signature Managing auditing Viewing and 1 In the QuantStudio 12K Flex Software select Tools Security gt Settings printing a user 2 In the Security Settings dialog box select the Users or Roles tab unt 3 Click View Report 4 In the Report screen click tool bar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item 5 Click Print to print the report or click H Save to save the report electronically PDF Close the report Managing auditing Enabling disabli ng Use the Audit screen to control the auditing state enabled or disabled the events that auditing are audited and the reasons available to users when audit mode is set to Prompt or Required Auditing is disabled by default IMPORTANT If you disable security you inactivate audit functions No audit record is generated for the inactivation of audit and electronic signature functions when you disable security 1 Inthe QuantStudio 12K Flex Software select Tools gt
40. Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 247 C Safety Chemical safety Biological hazard safety General biohazard WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories www cdc gov biosafety publications index htm e Occupational Safety
41. Possible cause Action Background calibration failed One or more wells of the background plate produced spectra that exceed the maximum limit for the instrument 1 Repeat the calibration using the same background plate 2 If the calibration fails again repeat the calibration using a different background plate 3 If the calibration fails again determine the source of the contamination as explained in Identifying contamination on page 80 Instrument does not eject the background plate The adhesive cover may have adhered the plate to the heated cover within the instrument 1 Power off the QuantStudio 12K Flex Instrument 2 Wait for 15 minutes then power on the QuantStudio 12K Flex Instrument and eject the plate 3 If the plate does not eject power off and unplug the QuantStudio 12K Flex Instrument then open the access door 4 Wearing powder free gloves reach into the QuantStudio 12K Flex Instrument and remove the plate from the heated cover then close the access door 5 Perform a background calibration to confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a local Life Technologies Field Service Office sy20 g ajdues pueg Aewy pue ayeyg N M MNW Bunesqneg Z4aydeyg Bunooysayqnos Z N A oipn s ueng swas soig panddy WL aping J SN uONesSiuiWpy DUS 32ULUIJUIEW W JS S YJd aw Jeay X2
42. Rotates materials to either the portrait or landscape positions where mechanism the range of motion is 135 270 total Grip Consists of two sets of fingers that grip the consumable The fingers close to grasp a consumable and open to release it Robot tower vertical axis Moves the arm up and down 54 6 cm 21 5 in from 16 5 71 1 cm 6 5 28 in above the table Rotary axis Rotates the arm 340 around the base of the Twister Robot Mechanical stops prevent continuous rotation Fixed position barcode reader Scans the barcodes of consumables as they are loaded into the QuantStudio 12K Flex Instrument Base cover Removable cover that contains four access bolts which secure the Twister Robot to the Sciclone ALH 3000 base Racks Provides storage for PCR consumables before and after they are run by the QuantStudio 12K Flex Instrument one of three shown Power LED When lit indicates the Twister Robot is powered on Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 27 1 Getting Started QuantStudio 12K Flex System hardware Rear view Component Description A On Off switch Power switch for the Twister Robot where the states are on or off 0 Power port 100 240V port that provides power to the Twister Robot RS232 port Provides serial communication with the computer Fuse cover T
43. Select the QC tab then review the summary of wells that failed the quality check QC Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 57 Calibrating Multi Well Plate and Array Card Sample Blocks Dye calibration 9 After you inspect the dye spectra verify the status of the calibration e Analysis Status Indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or the data it collected is unusable e QC Status Indicates the quality of the calibration data where passed indicates that all wells produced data that passed the quality check and failed indicates that one or more wells produced dye spectra that differ significantly from the other wells on the plate Analysis ac Action status status Passed Passed 1 Click Next 2 Enter any comments you have in the Comments field click Finish then click Yes when prompted to save the results 3 Remove the plate or array card when the QuantStudio 12K Flex Instrument ejects the tray arm Passed Failed Troubleshoot the failed calibration as described in Table 5 Troubleshooting dye calibrations on page 75 Failed Failed Note You can accept a calibration that passes the Analysis Status check but fails the QC Status check We recommend using calibrations that yield passing results for both
44. Signature tab 3 Select or deselect Enable e Signature IMPORTANT Enabling the electronic signature feature can substantially increase the size of experiment eds or template edt files 4 Click Apply Settings Configuring the Use the Security Settings dialog box to add or remove electronic signature meanings meanings of the and to determine the data types to which they apply The e signature meanings are the electronic text that a user can select to describe a reason for an electronic signature sig natures The QuantStudio 12K Flex Software is installed with the following default meanings Default data types Electronic Signature definition Plate Thermocycler Analysis Analysis setup Protocol Protocol Results Reviewed and Approved Plate Set Up Yes Yes No No Reviewed and Approved Results Yes Yes Yes Yes Reviewed and Approved Template Yes Yes No No Adding a meaning 1 In the e Signature tab of the Security Settings dialog box click Add in the e Signature Meanings settings 150 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Configuring the electronic signature rights for user roles Selecting the actions that require signature Chapter 6 Security Audit and Electronic Signature Managing electronic signature 2 In the Create Meaning dialog box enter a description of the e Signature meaning then click OK 3 Select what data is signed for the selected meani
45. Status Indicates the quality of the calibration data where passed indicates that all OpenArray calibration cases produced data that passed the quality check and failed indicates that one or more cases produced dye spectra that vary significantly Analysis statue Action Passed 1 Click Next 2 Enter any comments you have in the Comments field click Finish then click Yes when prompted to save the results Failed Discard the OpenArray calibration cases then prepare and run replacement cases If the calibration fails again contact Life Technologies for further assistance 2 When the instrument door opens remove the OpenArray Plate Carrier from the instrument tray A WARNING PHYSICAL INJURY HAZARD During instrument operation the cases can reach 100 C Ensure the cases are at room temperature before removing 3 Discard the OpenArray Calibration Cases IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the plate remove the plate as explained in Troubleshooting on page 105 94 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 3 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance Verifying the instrument performance When to perform the RNase P experiment About the OpenArray Plate RNase P Kit Installation specification Guidelines for handling the OpenArray
46. System Maintenance and Administration User Guide Contents ODP ino Z 5 A a E sade a ced Ta E AR EE 71 Identifyinig CONtAMIMAUON sanesna eee ea e De PLE e ee eal See aa 80 S CHAPTER3 Calibrating OpenArray Plate Sample Blocks 81 Recommended calibration and maintenance 0 00 eee eee e eee e eee eee 82 About the OpenArray Calibration Plaque 0000s cece ccc e eee eee ees 83 Caring for the OpenArray Calibration Plaque 00 00 cece cece eee eee 83 Background calibration 00 c ec e eset eee e enna 84 Required materials R e geeti a RTE R eee dee abe Wu RL K KRE L eae 84 When to perform the calibration 000 e eee eee eee ee eee ees 84 About the background calibration data 000 cece cece teeta 84 Load the plague Nees tid tobe sth area cna die e A aE 85 Rotate the plaque cece a XR R RN Re a RT beeen R RR 0 85 Complete the calibration ax K r 0 cece eee eee a R nee AK a enna 86 Uniformity calibration 23 2 Z nr 4224 TE lai 25 ade betes a ae Ale ot ce teh i ok 87 Required materials een mae ard hele rad REE een ee note eae 87 When to perform the calibration ccc eee eee eee eens 87 About the uniformity calibration 0c ccc ec e ee eeee eee ees 87 Load the plaque c eis 6 ie TET N bee OR eee eA PER ees ee es LR ee 88 Rotate the plaque ss ice bce peg eee eee vee eee ee eee ete R tG Peet R 09 K 88 Complete the calibration 0 000 c eee cette eee eee eee 89 DyercalibhatloncAcawn
47. USB 1 Component Description A Ethernet port An RJ45 port that provides Ethernet Gigabit communication with the QuantStudio 12K Flex Instrument t B USB ports Provide USB communication with the QuantStudio 12K Flex Instrument They can be used to transfer data to from the instrument and to update the firmware Note If multiple USB drives are plugged into the QuantStudio 12K Flex Instrument the instrument mounts only the first drive that is installed regardless of the USB port used C RS232 port Provides serial communication between the QuantStudio 12K Flex Instrument and the computer IMPORTANT The serial port is reserved for Life Technologies use only D Instrument fans Cool the interior of the QuantStudio 12K Flex Instrument IMPORTANT The fans must be unobstructed to ensure adequate cooling and proper function of the QuantStudio 12K Flex Instrument E On Off switch Power switch for the QuantStudio 12K Flex Instrument where the states are on 1 or off 0 F Fuse cover Dual 12 5A Time Lag T 250VAC 5 x 20 mm electrical fuses that protect the QuantStudio 12K Flex Instrument from excessive electrical current G Power port The 100 240VAC port that provides power to the QuantStudio 12K Flex Instrument T Use the Ethernet cable supplied with the QuantStudio 12K Flex System to connect the QuantStudio 12K Fle
48. Within 8 hours keep the ROI calibration plate at room temperature The ROI calibration plate is used in the uniformity calibration e After 8 hours return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate is stored in its original packaging sleeve you can use it to calibrate a QuantStudio 12K Flex System 3 times for up to 6 months after you open it 10 In the ROI Calibration screen click Finish to complete the calibration then click Yes when prompted to save the results 44 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Background calibration Background calibration When to perform the calibration About the background calibration data Preparing the calibration plate or array card During a background calibration the QuantStudio 12K Flex System e Performs reads of a background plate containing PCR buffer for 10 minutes at 60 C e Averages the spectra recorded during the run and extracts the resulting spectral component to a calibration file The QuantStudio 12K Flex Software then uses the calibration file during subsequent runs to remove background fluorescence from the run data Perform the background calibration monthly or as often as necessary depending on instrument use TM During the background calibration the Qua
49. allow the bottom of the RNase P plate to become dirty Fluids and other contaminants that adhere to the bottom of the reaction plate can contaminate the sample block and cause an abnormally high background signal Correct Incorrect a2 Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time Preparing an array IMPORTANT Perform the following procedure only if you are verifying the cardforinstrument Performance of a QuantStudio 12K Flex System with an array card sample block verification Required materials e Applied Biosystems Array Card Staker Sealer e Centrifuge with centrifuge array card carrier clips and buckets e Powder free gloves e Safety goggles e Pipettor 200 uL with pipette tips e TaqMan RNase P Array Card Instrument Verification Reagents Kit Array Card TaqMan RNase P Array Card Instrument Verification Reagents Kit including tubes with reagent mix for each port 8 tubes total Preparing the TaqMan RNase P Array Card IMPORTANT Wear powder free gloves while preparing the array card 1 Remove the Array Card RNase P Kit from the freezer then allow it to thaw at room temperature 2 Remove an array card from its box and place it on a clean dry surface 66 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks
50. an Applied Biosystems QuantStudio 12K Flex Real Time PCR System We recommend performing an RNase P instrument verification experiment e After moving the QuantStudio 12K Flex Instrument to another location e As needed to verify the function of the QuantStudio 12K Flex System The instrument verification experiment uses one of two instrument verification kits available from Life Technologies The kits differ only in the consumable format for which they are designed a TaqMan RNase P Instrument Verification Plate for QuantStudio 12K Flex Instruments with 96 384 well sample blocks and an Array Card RNase P Kit for QuantStudio 12K Flex Instruments with array card sample blocks TaqMan RNase P Instrument Verification Plates The RNase P plate is preloaded with the reagents necessary for the detection and quantitation of genomic copies of the human RNase P gene a single copy gene encoding the RNase moiety of the RNase P enzyme Each well contains TaqMan Fast Universal PCR Master Mix RNase P primers FAM dye labeled probe and a known concentration of human genomic DNA template The figure to the right illustrates the arrangement of the standard and unknown populations on a 96 well and Fast 96 well RNase P plate The RNase P plate contains five replicate groups of standards 1250 2500 5000 10000 and 20000 copies two unknown populations 5 000 and 10 000 copies and a no template control NTC Unknown A 50
51. and Administration User Guide Calibration Consumable Preparation This appendix covers Creating a background plate or array card 6 eee eee 182 Creating a custom dye plate for calibration 0 c cece eee eee 184 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 181 Calibration Consumable Preparation Creating a background plate or array card Creating a background plate or array card Whenever possible use a Background Plate or the TaqMan Array Background Buffer that is included with the spectral calibration kit The plates array cards supplied in the kit contain a buffer that accurately simulates the reagents used for PCR and therefore produces high quality calibration data If a background plate or array card froma spectral calibration kit is not available you can create one as described below Required materials 96 384 Well Plate Sample Block Applied Biosystems Optical 96 384 Well Reaction Plate Safety glasses Optical Adhesive Cover or Optical Flat Caps Pipettor 200 uL with pipette tips Powder free gloves Deionized water Array Card Sample Block Array Cards Applied Biosystems Array Card Staker Sealer Centrifuge with array card buckets and array card carrier clips Permanent marker or pen Pipettor 200 uL with pipette tips Powder free gloves Safety glasses Deionized water Creating a IMPORTANT Wear powder free gloves while cr
52. and Administration User Guide Preparing the calibration plate or array card Preparing the calibration plates Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Dye calibration Prepare the Dye calibration consumables appropriate for your QuantStudio 12K Flex Instrument S Preparing the dye calibration plate 0 eee 55 E Preparing array cards for instrument calibration lt e ran 37 IMPORTANT Before performing a dye calibration you must perform an ROI calibration a background calibration and a uniformity calibration IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or array cards Required materials e 96 or 384 Well Spectral Calibration Plates FAM Dye VIC Dye ROX Dye NED Dye TAMRA Dye and SYBR Green Dye e Centrifuge with plate adapter e Powder free gloves e Safety goggles Preparing the dye calibration plate 1 Remove the dye plates from the freezer then allow them to warm to room temperature approximately 5 minutes IMPORTANT Do not remove the dye plates from their packaging until you are ready to run them The dyes in the dye plates are photosensitive Prolonged exposure to light can diminish the fluorescence signal strength of the plates Note If you store dye plates frozen and in their original packaging you can use them to calibrate a QuantStudio 12K Flex System up to 3 times for 6 months after opening
53. and files 20 00005 117 Managing licenses ccs ce et dd S E N en ae eter E taal st ee Dana Ace atari te ae 117 Replacing the sample bock ccc eee eee eee eee 119 Required materials 0c cece ccc ee eee eee e eee e eae 119 Handling the sample block 2 acee0est beatae ehieee eee bie ARR h R a R 119 Replacing the sample bock 119 Replacing the heated cover ccc cece eee eee eee e eee aee 121 Required materials 0c cece ccc ee eee ence eee e eens 121 Handling the heated cover 0 cece cect rnrn rreren e eae 121 Replacing the heated cover 20 cee cece e eet nett e eee eenaes 121 Replacing the plate adapter 020 00 nannan narenn eee eee e eens 123 Requiredinaterialss oni scotia sate seg pee te alec Ri oll woh N ea lees 123 Replacing the plate adapter 0 0 6 cnet tenet eee nas 123 R CHAPTER 5 Networking iicstsc 504 e x e K K K odes nee tetans 125 Networking overview es c ccb eed gee heen Gables Vokes Gadled pulndauees ea ddet ee tet ne 126 Controlling and monitoring networked Instruments eee cece eens 126 About the Ethernet part ccc ig beac kg eit adie ide ol de hdd A Ed aT 126 Example network layouts cece eee eee eee e eee e eee eee 127 Networking guidelines and best practices 0c c cece eee eee eee 128 Network Setup Workflow lt esep a T SRL cee eee ee ee ea ea LA 128 Collecting the required network Information cece eee eee ee eeeeee 129 Connecting the QuantSt
54. ates EEE EE E elas 230 E Accessories n iene a E ed Pde Soke es a ae dea aes 232 E Calibration and verification MS 0 0 c ccc cence 233 R Consumables jac esc gassed oe als ned Raia me ene E Dare BOs aah 236 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 229 Parts and Materials How to order How to order You can order materials and accessories from Life Technologies by ordering directly from the Life Technologies online store Note Product availability and pricing may vary according to your region or country Online ordering through Life Technologies is not available in all countries Contact your local Life Technologies representative for help To order through the website or the QuantStudio 12K Flex Software e Confirm that your computer has an Internet connection e We recommend the following browsers and Adobe Acrobat Reader versions to use the Life Technologies website i Microsoft Apple Mozilla Adobe P Operating system Internet Safari Firefox Acrobat Explorer Reader Microsoft Windows v x or later Nonet v2 x or later v4 0 or later Macintosh Nonet v2 0 4 or later T Browser not available for this platform Note Confirm that cookies and Javascript are turned on for the website to function correctly 230 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance
55. be monitored by more than 10 computers simultaneously s Unless you are sure that your QuantStudio 12K Flex Instrument and computer exist on the same subnet we recommend that you use the IP address of the QuantStudio 12K Flex Instrument to add it for remote monitoring Monitoring the 1 In the Home screen of the QuantStudio 12K Flex Software click Instrument status of an Console instrument during 2 In the Instrument Console select your QuantStudio 12K Flex Instrument from y a run the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it 3 After the instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager click Monitor then click Information 5 In the Monitor Instrument screen click Monitor Running Experiment The QuantStudio 12K Flex Software displays the status attributes calibration status and plot data for the selected QuantStudio 12K Flex System If a communications warning appears contact your network administrator to troubleshoot the problem You can lose the software connection to the QuantStudio 12K Flex Instrument if you e Change the QuantStudio 12K Flex Instrument that is connected directly to your computer e Use the touchscreen to change the instrument name or IP address Note To reestablish the connection resta
56. by the assay in lt 100 character string gt the associated well S Target Color Optional The RGB color of the target RGB lt r gt lt g gt lt b gt 8 E Task The task assignment of the target assay at the wel TT lt UNKNOWN STANDARD S NTC ENDOGENOUS S IPC BlockedIPC gt T Reporter The reporter dye used by the associated target assay lt dye name gt 88 x Quencher The quencher dye used by the associated target assay lt dye name gt 88 s Quantity Optional The quantity of standard present in the given well lt float or integer gt expressed as a float or integer If the associated well is not assigned the STANDARD task then the field is blank SNP Assay Name The name of the SNP assay detected or amplified by the assay lt 100 character string gt in the associated well SNP Assay Color Optional SNP assay color in RGB RGB lt r gt lt g gt lt b gt 8 Task The task assignment of the SNP assay at the well lt UNKNOWN NTC PC_ALLELE_1 PC_ALLELE_2 gt PC_ALLELE_BOTH gt Allele1 Name The name of the first allele detected by the SNP assay lt 100 character string gt G Allele1 Color The RGB color used to represent data for the first allele RGB lt r gt lt g gt lt b gt nB Allele Reporter The reporter dye used to label the probe for the first allele lt dye name gt 8 v Allele1 Quencher The quencher dye used to label the probe for the first allele lt dye name gt 8 amp
57. card pipet the entire 100 uL volume into the fill reservoir but do not go past the first stop of pipettor plunger or you may blow the solution out of the port IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port 5 Repeat step 4 to fill the remaining array card with the appropriate calibration reagents carrier clip and place empty array cards in the remaining slots Confirm that the labels on the buckets and clips face the same way Make sure that the array card fill reservoirs and bucket and clip labels face outward when loaded into the centrifuge IMPORTANT You must run the centrifuge with all four buckets in place and each of the two carriers filled with array cards Place empty array card into unfilled slots IMPORTANT Balance the loads in opposite buckets in the centrifuge 8 Close the centrifuge cover then spin the array card s for 1 minute at 1200 rpm Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Preparing array cards for instrument calibration 9 When the run is finished stop the centrifuge then spin the array card s again for 1 minute at 1200 rpm IMPORTANT Do not try to save time by doing one spin for 2 minutes The two sets of ramps are important for a good fill into the array card 10 When the sec
58. cata E 9 a Ne Best Ait hes A hod oe Fe tek a 90 Required materials 0c cect eee eee nese eee e eee aee 90 When to perform the dye calibrations 00 cece eee eee eens 90 About the dye calibration 0 0 c cece eee eee eect eee eee eeeees 91 Guidelines for handling the OpenArray Calibration Cases 2 00 c cece eee eee 91 Perform the empty reading 0 cc cece cee eee eee eee ee ee 91 Perform the filled reading n 02 cee cece eee eee eee e eee e eae 92 Complete the calibration 0 000 cece cect teen eee eee 94 Verifying the instrument performance 20000 cece eee cece eee ee nrnna 95 When to perform the RNase P experiment cece cece cece tee eeees 95 About the OpenArray Plate RNase PKL 95 Installation Specification 24 00 Te ZEE K cap ea child re Eola ele 95 Guidelines for handling the OpenArray plate 95 Required materials v 00 sc 2cce ree ev twee rede ee See eho wae te ye he NR T be Seabee onal 96 Preparing for the verification experiment 00 ec e cece cece e ee eens 96 Initializing the system 00 c cece eeee eens 97 Preparing 0G lading 3 n a niet head Hit eps TR A ea ee ad Sole abel 98 Loading the OpenArray plate 2 0 0 0 cic cnn nee n ence eens 99 Sealing the OpenArray plate 0 occ ccc ene r nent rnrn neces 101 Running the experiment 2 0 00 c eect eee eee eee 103 Troubleshooting eeina heee Pale naira Gable hayes pe ed dep eres eee dod Reeders 105 Identifyi
59. ccc eee e eect teens 183 Creating a custom dye plate for calibration arannana rnnr rnnr 184 Before you use custom dyes 2 0 cece ccc eee eens 184 Required materials bee eve hey eae ee PoE Pepe ode e Feed bade vem ee oebve eee 184 Determining optimum dye concentration cece eee eee 184 Creating a custom dye plate 0 0 0 cece cece ttt ee eeeeee 185 Adding the custom dye to the software 0 00 cece eee eee teens 186 APPENDIX D Command line Software Operation 189 OVERVICW ns T Net te tl E Sh ial Ge SE tana Meaghan ty MN Dart at 190 Command line workflows 0000 cece cece cece cette eet ee teenies 190 Supporting files for experiment creation 0 0 ccc cece eect eet eeeeee 191 Precedence rules for experiment file generation cece eee cece eee 192 Running the command line application 000s 193 Running the application 000 cence eens 193 Viewing the command line help 0c c cece cece cece ee eee ees 193 Command syntax and arguments nannan nannan rnrn nrnna e eee e eee aee 194 Batch tile creation e peia beat tl bay TT 194 RESULTS export narri anni da a N s Pek d dete a aak etree eit daha Sc diaa 196 Examples an nra t te kev Reee rele E en meine ede ne tatedde ne Selden eran Re 197 Batchitile Creation a RE eL RT Se een see ha the ae ad lala 197 Results CX POT A Zoettaae lane R N R 2 re ah eae E 197 APPENDIX E File Format Reference see 199 Import format
60. click Save or e Click gh Print Click OK in the confirmation message Exporting and importing settings Note The export import feature can be used to replicate identical security audit and e signature settings across multiple computers The feature allows you to create a standard security audit and e signature settings image for the QuantStudio 12K Flex Software that can then be imported by other copies of the software to bypass manual setup Exporting settings 1 2 Importing settings 1 In any screen of the Security Settings dialog box click Export Select the items to export e All Contains all settings e Custom Contains select settings Users amp Roles All user accounts with Active status and all user roles and associated permissions in case a user account specifies a user role that does not exist on the system into which you import the profiles System amp Roles All system settings and all user roles and associated permissions Click Export or OK When prompted specify the name and location for the exported file dat then click Save A message is displayed when the export completes In any screen in the Security Settings dialog box click Import in the navigation pane Select the dat file to import then click Open A message is displayed asking if you want to overwrite the current system configuration Click Yes If any imported user accounts already exist o
61. e Absent The sample block is contaminated Decontaminate the sample block again and repeat the calibration Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 107 Calibrating OpenArray Plate Sample Blocks Troubleshooting Viewing the ROX You can export quality control QC images from RNase P experiments The QC image files images include calibration images a barcode image and images taken during the run You can view the images to check that calibration was correct or to validate the data To export QC images from an RNase P experiment 1 In the Instrument Manager click Export while viewing the results of the RNase P run 2 In the Export screen select a location for the file Click Browse if you do not want to save the image files to the default export folder Note To set up the Export File Location go to Tools gt Preferences select the Export tab then select Use Last File Location or Use Default Folder If you do not specify a directory to receive the image files the software exports the files to the default directory C Applied Biosystems QuantStudio user files experiment s 3 Click Export QC Images 4 Using a graphics editor program such as Microsoft Paint open and review each QC image Problem symptom Possible cause Action No amplification in the OpenArray case is CAUTION PHYSICAL through holes along the long sealed improperly INJURY HAZ
62. edt The values in the template take precedence except for s Experiment Name Determined by the File Name Convention preference e Barcode Determined by the barcode if present Otherwise the value is null e Experiment File Name Determined by the File Name Convention preference s Template file edt e Assay information file aif aix All values in the template file take precedence except for e Gene Expression Targets Assay Definition e Genotyping Assay SNP Definition e Passive Reference If any conflicts exist between the assay information file and the template for the attributes above then the assay information file values always take precedence e Template file edt e Plate setup file txt All values in the template file take precedence except for e Target Assay SNP to Well Assignment e Biological Group Color e Sample to Well Assignment e Target Color e Task to Well Assignment e Gene Expression Targets Definition e Biological Group to Well Assignment e Genotyping Assay Definition e Well Quantity to Well Assignment e Passive Reference e Sample Color s Template file edt e Plate setup file txt e Assay information file aif aix All values in the template take precedence except for the following The following assay information file values take precedence over Plate Setup and Template e Gene Expression Targets Detectors Definition e GT Assay Marker Definition e Pass
63. eee eens 123 IMPORTANT This chapter contains all user service procedures for the Applied Biosystems QuantStudio 12K Flex Real Time PCR System Procedures other than those described in this document must be performed by a qualified Life Technologies service engineer Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 109 4 Maintenance Regular data maintenance Regular data maintenance Maintaining the computer hard drives Archiving and backing up experiment files Backing up the instrument settings Defragment and clean up the hard drive e Atleast once every month e When a message is displayed by the Windows operating system instructing you to defragment For more information on maintaining the hard drives see the Windows Operating System Help then search the Help to find information on the Disk Cleanup and Disk Defragment utilities IMPORTANT Do not run the disk management utilities and QuantStudio 12K Flex Software at the same time Archive experiment files regularly To conserve space on the computer hard drive older EDS files can be archived using a data compression utility Several commercial compression utilities are available to store experiment files in the ZIP or ARC archive format Back up experiment files We strongly recommend that you back up your experiments Backing up data e Protects against potential loss of data caused by fa
64. experiment runs to characterize and distinguish the individual contribution of each dye in the total fluorescence collected by the instrument In the software a collection of dyes to use in experiments Custom dyes can be added to the library but system dyes cannot be removed Before using a dye make sure that the dye calibration is current in the Instrument Console See amplification efficiency EFF In Comparative Cy experiments a feature that allows you to manually enter previously determined amplification efficiencies for each experiment following the experimental run The real time software mathematically compensates for differences in efficiency between each target assay and the endogenous control when calculating sample to sample relative quantities This method can be employed as a substitute for the Relative Standard Curve Method 256 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide endogenous control endpoint read error experiment experiment document experiment name Experiment Setup experiment type export Glossary A gene that is used to normalize template differences and sample to sample or run to run variation See post PCR read The standard error of the slope of the regression line in the standard curve The error can be used to calculate a confidence interval CI for the slope Because the amplification efficiency EFF is calcula
65. extreme cases influence experimental results IMPORTANT Do not use organic solvents to clean the QuantStudio 12K Flex System Table 1 Multi well plate and array card sample block maintenance Frequency Maintenance task Weekly Check the computer disk space If necessary archive or back up your experiment files and instrument settings Power off the computer that controls the QuantStudio 12K Flex System then after 30 seconds power on the computer Clean the surface of the QuantStudio 12K Flex System with a lint free cloth Perform a QuantStudio 12K Flex Instrument self test Monthly Perform a background calibration T Run disk cleanup and disk defragmentation Annually Perform a regions of interest ROI calibration Perform a background calibration Perform a uniformity calibration Perform a dye calibration Perform a normalization calibration Perform an instrument verification run As needed Decontaminate the QuantStudio 12K Flex System Replace the QuantStudio 12K Flex System fuses Update the Windows operating system Update the QuantStudio 12K Flex Software and firmware T You can perform a background calibration to check for contamination If any parts of the optics are replaced or moved you must perform all calibrations including an RNase P instrument verification run 36 Applied Biosystems QuantStudio
66. field to enter a reaction volume in uL e Optional Touch the Barcode Number field to enter a barcode and touch the Notes field to enter notes up to 200 characters about the experiment 6 When finished either e Touch gm Save amp Exit or e Touch Save amp Start Run to proceed to the Start Run screen Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 159 A Manual Instrument Operation Operating the instrument from the touchscreen Running an 1 If the instrument is in standby touch the QuantStudio 12K Flex Instrument experiment touchscreen to activate it then press 4 2 In the Main Menu screen then touch E 3 When the side door opens load the appropriate plate or array card Ensure that the consumable is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the barcode facing the front of the instrument 4 In the Main Menu touch Browse Experiments 5 In the Experiments screen touch the desired experiment then touch either N Start Run to start the run immediately then go to step 10 or e G View Edit to view or edit the experiment before starting the run 6 Modify the experiment parameters as needed To e Adda stage or step to the thermal profile touch the stage or step to the left of where you want to add the stage or st
67. firmware update transfer the update to the QuantStudio 12K Flex Instrument using a USB drive Updating the firmware 1 Download the firmware update a Go to www lifetechnologies com support software b In the Software Downloads page select Applied Biosystems QuantStudio 12K Flex Software from the menu c In the Software Downloads page for your QuantStudio 12K Flex Instrument click Updates Patches d Download the QuantStudio 12K Flex Instrument firmware to a USB drive 2 Plug the drive into the USB port on the right side of the QuantStudio 12K Flex Instrument touchscreen 3 If the instrument is in standby touch the QuantStudio 12K Flex Instrument touchscreen to activate it then press 0 4 In the Main Menu touch Tools then touch g Upgrade Firmware 5 In the Upgrade Firmware screen select the update package then touch Upgrade Firmware Allow the QuantStudio 12K Flex Instrument to complete the upgrade IMPORTANT Do not remove the USB drive from the QuantStudio 12K Flex Instrument until you are instructed to do so 6 After the upgrade is complete and the QuantStudio 12K Flex Instrument reboots confirm the upgrade success a Unplug the USB drive b Touch Settings then touch About this instrument to view the software version number to confirm that the firmware has been upgraded Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Adminis
68. for a cycling stage the settings are indicated by an icon in the thermal profile e AutoDelta on A e AutoDelta off A An analysis setting in which the software calculates the baseline start and end cycles for the amplification plot See also baseline An analysis setting in which the software calculates the baseline start and end cycles and the threshold in the amplification plot The software uses the baseline and threshold to calculate the threshold cycle Cy See also threshold cycle CT Type of calibration in which the instrument performs reads of a background plate averages the spectra recorded during the run and extracts the resulting spectral component to a calibration file The software then uses the calibration file during subsequent runs to remove the background fluorescence from the run data In the amplification plot a cycle to cycle range that defines background fluorescence This range can be set manually on an assay by assay basis or automatically to set each individual well 254 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide baseline corrected normalized reporter ARn baseline threshold algorithm biological replicates blocked IPC calibrator chemistry comparative Cy AAC method CRT Car algorithm Cr Glossary The magnitude of normalized fluorescence signal generated by the reporter In experiments that contain data from r
69. for the sample at the well Float Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 227 E File Format Reference RDML export format Genotyping experiments The following table describes the results data exported from genotyping experiments Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 character string SNP Assay The name of the SNP assay added to the well 100 character string Name Allele1 Rn The raw fluorescence associated with the allele 1 Float probe of the SNP assay at the well normalized to the fluorescence of the passive reference dye Allele2 Rn The raw fluorescence associated with the allele 2 Float probe of the SNP assay at the well normalized to the fluorescence of the passive reference dye Call The allele call assigned to the sample at the Homozygous specified well lt allele x allele x gt Heterozygous lt allele x allele y gt or Negative Control NC Quality The confidence of the automatic allele call Float 1 100 Method The method used to call alleles Auto Or Manual Task The task assigned to the target in the well UNKNOWN or NTC Pass Ref The raw fluorescence of passive reference
70. local Life Technologies Field Service Office k Bunooysayqnos s4901g ajdwes pieg Aewy pue 3 eld N M MNW Bunesqneg olpnysjueng swaj stsoig panddy wl aping Jasf uonensiuIUpy pue aueuajulew W IS S YJd awl jeay Xa4 YZ LL Table 7 Troubleshooting RNase P instrument verification experiments Problem symptom Possible cause Action More than the maximum number of outliers are present in RNase P data Possible contamination Pipetting inaccuracy Contact Life Technologies to order a replacement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Life Technologies for further assistance RNase P plate verification run failed Insufficient centrifugation CAUTION PHYSICAL INJURY HAZARD During instrument operation the sample block can be heated to 100 C Before performing the following procedure wait until the sample block reaches room temperature 1 Unload the RNase P plate or array card from the QuantStudio 12K Flex Instrument 2 Hold the plate or array card up to a light source to verify that all wells contain the same volume of fluid 3 If there are differences in fluid volumes check the heat seal of the wells with lower Defective plate seal volumes for signs of damage or evaporation Compare the position of the wells that have lower volumes with the outliers that you have removed from the plate If the well positions coincid
71. must contain all the elements described in the following section and in the order that they appear Note The command line application see page 189 does not import sample files If you are using the application to create experiments use plate setup files to import sample information into the new experiments see Plate setup file format on page 201 Sample file header row The sample file begins with an optional header row that contains column headers for well number Well sample name Sample Name and optional custom properties names The order of the columns is important and cannot be changed Sample file body A body of rows containing the sample data follows the optional header row Each body row defines the sample information for a single well on the consumable including well number sample name and any applicable custom fields The body can contain data for a subset of wells on the consumable so the rows for empty wells can be omitted from the file The sample body rows can occur in any order Couumn Description Valid values name Well The number of the well on the consumable lt Positive integer 1 96 where the well numbers start at 1 for well A1 384 gt upper left corner and increase from left to right and from top to bottom Sample The name of the sample contained by the lt 100 character string gt Name associated well Custom1 Optional Additional text that describes th
72. nn 177 Storing the QuantStudio 12K Flex System 0 cece eee eee 178 Moving the QuantStudio 12K Flex System 0 6 660s cece eee 179 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 175 Powering On or Off Storing and Moving the System Placing the QuantStudio 12K Flex System on standby Placing the QuantStudio 12K Flex System on standby If left unattended the QuantStudio 12K Flex Instrument automatically enters standby mode to conserve power To enter standby mode manually touch GJ on the QuantStudio 12K Flex Instrument touchscreen Powering on the QuantStudio 12K Flex System To power on the QuantStudio 12K Flex System from a powered off state 1 Toggle the power button on the rear of the QuantStudio 12K Flex Instrument then wait for it to start Note The QuantStudio 12K Flex Instrument is ready to use when the touchscreen displays the Main Menu 2 If you have an Applied Biosystems Twister Robot toggle the power button on the rear of the Twister Robot Note The Twister Robot is ready to use when the power LED illuminates 3 Power on the monitor 4 Power on the QuantStudio 12K Flex System computer a Press the power button of the computer then wait for it to start b When the Login screen appears enter your user name and password then click OK c In the desktop double click QuantStudio 12K Flex Syste
73. off the monitor 4 If you have an Applied Biosystems Twister Robot toggle the power button on the rear of the Twister Robot Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 177 Powering On or Off Storing and Moving the System Storing the QuantStudio 12K Flex System Storing the QuantStudio 12K Flex System Required materials Preparing the QuantStudio 12K Flex Instrument The Applied Biosystems QuantStudio 12K Flex Real Time PCR System can be powered off and stored for extended periods of time The length of the period of inactivity determines the method you use to power off the QuantStudio 12K Flex Instrument MicroAmp Optical 96 384 Well Reaction Plate or array card unused 1 If you plan to store the QuantStudio 12K Flex System for more than a week or you plan to move it load an unused plate or array card into the QuantStudio 12K Flex Instrument Note The empty plate protects the internal components of the QuantStudio 12K Flex System during transport or during periods of inactivity lasting more than a week a Touch the QuantStudio 12K Flex Instrument touchscreen to activate it then touch g b Touch to eject the tray arm place a plate or array card onto the plate adapter then press ES again to load the plate c Touch 0 to place the QuantStudio 12K Flex Instrument into stand by mode Toggle the power button o
74. operation For example the folder created for files generated on April 7 2010 at 12 48 35 would be 2010 04 07 124835 Results export The following example performs a real time data markup language RDML export of experiments in the QuantStudio 12K Flex Software experiments directory to the exports directory of the C drive The software generates an RDML file for each individual experiment file cmdlineutil ex xport C Applied Biosystems QuantStudio 12K Flex Software User Files experiments f SDS23 1 c exports s single x rdml Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 197 D Command line Software Operation Examples 198 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide File Format Reference This appendix covers Import formats and file specifications 0 0 0 6 cece eee eee 200 Plate setup file format 0 be his doled tea dhe wai R 201 Sample file format sisendis riiai sa die hag ite ha Galt ete ba sheila 206 Barcod file formate rere roaster te E RRR ATR wet b edna eet Sale ata 207 Assaysinformation Messie e Get cee got lk aes eb dig ghee eshte be ghia eee 207 Export formats and file specifications 066 6 cece cece eee 208 QuantStudiol2KFlex export format e eee eee eee 209 7200 export format ase ihe et atecs PASSA ee SS ee Rn Sela eh 223 RDMLvexpo
75. or edit 142 delete 142 inactivate 142 permissions 143 user role create 143 V verification array cards 37 66 consumables 236 kits 233 OpenArray plate 96 plate 65 VIC dye 19 53 W WARNING description 14 waste disposal guidelines 247 waste profiles description 247 weekly maintenance tasks 36 82 weight instrument 20 workstation safety 243 Y y intercept 268 278 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 6890477 Headquarters L 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support technologies www lifetechnologies com
76. plate IMPORTANT Perform the following procedure only if you are performing a verification experiment for a QuantStudio 12K Flex System with an OpenArray plate sample block IMPORTANT When performing the RNase P instrument verification experiment e Perform all calibrations first s Run the OpenArray plate soon after you allow the plate or reagents to thaw Minimizing the time between thaw and run ensures optimal performance e Wear powder free gloves and safety glasses when you prepare OpenArray plates Perform the RNase P instrument verification experiment to verify the performance of the Applied Biosystems QuantStudio 12K Flex Real Time PCR System We recommend performing an RNase P instrument verification experiment e After moving the QuantStudio 12K Flex Instrument to another location s As needed to verify the function of the QuantStudio 12K Flex System The RNase P Kit includes one empty OpenArray plate and a single tube that contains OpenArray RNase P Reaction Mix TaqMan Universal PCR Master Mix RNase P primers and FAM MGB dye labeled probe and a known concentration of human genomic DNA template The QuantStudio 12K Flex System passes the installation specification if the standard deviation of the Cy values for all through holes on the OpenArray plate is lt 0 25 The data from up to 48 through holes can be omitted from the population to meet the installation specification e Hold th
77. protocol run results analysis protocol and other plate specific data plate setup file LH A user created tab delimited text file that describes the layout of a consumable for an experiment to be run on the QuantStudio 12K Flex System The file defines the arrangement of assays and samples on the consumable See Plate setup file format on page 201 for more information sample file txt A user created tab delimited text file containing sample data that can be imported into the QuantStudio 12K Flex Software for use in related experiments See Sample file format on page 206 for more information Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 191 D Command line Software Operation Precedence rules for experiment file generation Precedence rules for experiment file generation When generating experiment files eds the QuantStudio 12K Flex Software command line interface relies on a set of precedence rules to resolve conflicts that arise from the data supplied by some input files Assay information files aif or aix plate setup files txt and template files edt can contain data used to populate the same fields of new experiment files For example both template and plate setup files can contain location data for samples and assays Files used for experiment file eds creation Precedence rule Template file
78. pulling the enclosure door up IMPORTANT To safely operate the instrument keep the deck clear and have enough room in the waste bin to eject the used pipette tips b Empty the waste bin and place it back on the deck Waste bin Plate holder 2 Remove any OpenArray plates from the deck Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 97 Preparing for loading 98 3 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance If necessary replace the tip boxes Note Tip boxes contain 384 tips divided into 8 sections When you click Load the OpenArray Accufill instrument loads as though a new full box of tips is on the deck OpenArray Accufill software prompts you to verify that tips are in the locations shown in the Setup Deck screen Clicking a section in the Setup Deck window confirms that tips are in that section of the tip box We recommend using a full tip box a Place tip boxes into the assigned locations b Place tip boxes on the deck in the two side by side recessed rectangular platforms c Remove the cover before using the tips for loading Close the door on the instrument Click Proceed to begin the System Self Test The application performs a number of self tests and is then ready for you to continue Note System Self Test runs only at start up The test does not run again unless the system is res
79. results instrument information and security and auditing settings to spreadsheet presentation or text files You can edit the default location of the exported file Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 257 Glossary filter flag forward primer genotyping experiment heterozygote holding stage homozygote housekeeping gene import Instrument Console Instrument Manager 258 Dye excitation and emission filter combination that you select for an experiment The QuantStudio 12K Flex System includes a six color filter set that supports FAM NED ROX SYBR Green TAMRA and VIC dyes A quality control QC indicator which when applied by the software to a well during analysis indicates a possible issue with that reaction For example a flag may be issued if no amplification is detected in a well Flags indicating potential problems are displayed in the Quality Control tab of the plate layout well table and QC Summary screens Oligonucleotide that flanks the 5 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target An experiment used to identify known mutations in a DNA sample With this experiment type you can determine if a DNA sample is e Homozygous samples having only allele 1 Also called wild type homozygote e Homozygous samples having only allele
80. signs the message is displayed again Generating electronic signature reports Displaying electronic signature records Saving or printing electronic signature records You can use the QuantStudio 12K Flex Software to generate reports of e signature history from open experiment eds or template edt files 1 Open an experiment eds or template edt file 2 In the open experiment or template click o Audit then click E Signatures 3 Optional Click any row to view details for individual signatures 1 Open an experiment eds or template edt file In the open experiment or template click o Audit then click E Signatures In the table select the record to be saved or printed La T Save or print the record e Click IS Save select a location for the export file enter a name for the file then click Save or e Click 5 Print 5 Click OK in the confirmation message 152 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Saving or printing 1 the table of 2 electronic signature events s 4 Chapter 6 Security Audit and Electronic Signature 6 Exporting and importing settings Open an experiment eds or template edt file In the open experiment or template click ve Audit then click Print E Signatures Save or print the record e Click Save select a location for the export file enter a name for the file then
81. starts with header an asterisk and ends with a carriage return in the following pattern lt field name gt lt field value gt The header must contain the lines shown in the following table Field Description Valid values Instrument The model of QuantStudio 12K Flex QuantStudio12KFlex Type System for which the experiment is designed Passive The dye that the experiment will use e The name of a dye in the Dye Reference as a passive reference Library of the QuantStudio 12K Flex Softwaret or e lt blank gt if the consumable does not contain a passive reference T Custom dyes are allowed if they are in Dye Library Note The QuantStudio 12K Flex Software automatically removes any leading and trailing white space around the field name and field value Example Instrument Type QuantStudiol2KFlex Passive Reference ROX Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 201 E File Format Reference Plate setup file format Plate setup file body The body of a plate setup file contains either target information which can be imported into all experiments except genotyping or SNP assay information This information can be imported into genotyping experiments only The body consists of three required elements the header the column header and the body that describe the contents of a 96 384 well plate or array card The sample
82. text files are supported The following table shows the data files that the QuantStudio 12K Flex Software can export in the 7900 export format Each row represents a single exportable data file File Description See Setup file Describes the configuration of samples on the experiment page 223 consumable including sample location target or SNP assay properties and task assignments Multicomponent Contains the spectral data used by the QuantStudio 12K page 225 file Flex Software to generate the multicomponent plot that displays the contribution of each dye over the duration of the PCR run Results file Contains the normalized processed and analyzed data page 226 generated by the QuantStudio 12K Flex Software TM When setup file is selected as an export option the QuantStudio 12K Flex Software exports sample setup data to a stand alone file The sample setup file describes the sample configuration on the experiment consumable including sample and assay data positions and task assignments The file begins with several lines shown in the following table that describe the experiment file and the QuantStudio 12K Flex Instrument for which it is designed Category Component Output File Version Defines the version of Setup File format used to Integer generate the document Plate Size Defines the number of wells in the plate Integer modeled by the file for example 96 384 Plate ID
83. the data used to generate the file multicomponent plot of a real time PCR experiment The multicomponent data tracks the raw fluorescence of all reporter dyes present on the reaction consumable throughout the duration of the experiment run The file begins with a line that names the export format SDS 2 3 and the type of data contained by the file multicomponent A column header occurs next followed by the multicomponent data where each row contains the data for a single well separated by tab characters The multicomponent data contains a dye column for each dye present on the reaction consumable including reporter dyes quencher dyes except non fluorescent dyes and the passive reference Column Description Output Well The number of the well on the consumable Integer 1 96 384 Time The time in milliseconds after the start of the Integer run when the reading was taken Temp The temperature C of the sample when the Integer QuantStudio 12K Flex Instrument recorded the fluorescence data Cycle The cycle of the run during which the Integer QuantStudio 12K Flex Instrument recorded the fluorescence data lt Dye name gt The raw fluorescence for the designated dye Float measured by the QuantStudio 12K Flex Instrument at the specified well and cycle Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems QuantStudi
84. the instrument icon The instrument icon is the graphic used to represent the QuantStudio 12K Flex Instrument in the QuantStudio 12K Flex Software Instrument Console a Save the replacement graphic to a USB drive then plug the drive into the USB port on the right side of the QuantStudio 12K Flex Instrument touchscreen b Touch Upload Icon select the desired graphic file then touch Done Note The replacement graphic must be a maximum of 48 x 48 pixels and be stored in the portable net graphic PNG format c Unplug the USB drive 5 Define the standby time out setting a Select Standby Time out to activate the feature b Touch the Standby Time out field Appendix A Manual Instrument Operation Administering the instrument from the touchscreen c Enter the number of minutes 1 300 that the QuantStudio 12K Flex Instrument should remain idle until it enters standby mode then touch Done Note When in standby mode the QuantStudio 12K Flex Instrument powers off the LCD screen backlight and enters low power mode 6 Define the heated cover temperature setting a Select Cover Idle Temperature to activate the feature b Touch the Cover Idle Temperature field c Enter the temperature 50 110 C that the heated cover should maintain when the QuantStudio 12K Flex Instrument is idle then touch Done 7 Touch Save to save the settings then touch OK when prompted 8 Touch Q to return to the
85. the normalization standards in a normalization calibration file The normalization calibration generates factors that the QuantStudio 12K Flex Software uses when comparing data from multiple QuantStudio 12K Flex Instruments Perform a normalization calibration every year or as often as necessary depending on instrument use During the normalization calibration the QuantStudio 12K Flex Software captures a series of images of each normalization plate using each instrument filter The normalization calibration yields a Pass or Fail result for each normalization plate used Prepare the calibration consumables appropriate for your QuantStudio 12K Flex Instrument m Preparing the normalization plates 59 E Preparing array cards for instrument calibration lt e sussns rnrn 37 IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or array cards IMPORTANT Before performing a normalization calibration you must perform ROL background uniformity and dye calibrations Required materials e 96 or 384 Well Normalization Plates with FAM ROX and VIC ROX Dyes e Centrifuge with plate adapter e Powder free gloves e Safety goggles Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 59 Calibrating Multi Well Plate and Array Card Sample Blocks Normalization calibration Performing the calibration 60 Prepari
86. the sample block into the QuantStudio 12K Flex Instrument then close the access door IMPORTANT After installing the sample block confirm that the indicator on the left side of the sample block is positioned behind the red line on the instrument rail If the indicator is forward of the red line push the sample block into the QuantStudio 12K Flex Instrument until it is seated correctly Close the access door IMPORTANT Confirm that the access door is completely closed The QuantStudio 12K Flex Software displays an error message if the door is not completely closed and latched or if the sample block is not seated correctly Plug in then power on the QuantStudio 12K Flex System Perform a background calibration to confirm that you have eliminated the contamination If the contamination remains repeat steps 2 through 5 then clean the contaminated wells of the sample block using a 95 ethanol solution a Pipet a small volume of 95 ethanol solution into each contaminated well b In each contaminated well pipet the solution up and down several times to rinse the well If you are decontaminating an array card or OpenArray plate sample block swab the surface of the block that contacts the consumable c Pipet the ethanol solution to a waste beaker Repeat steps 5 through 9 to rinse the wells of the sample block and to verify that you have eliminated the contamination IMPORTANT Always use deionized wat
87. to and restores instrument settings from a USB Flash Drive Before backing up or restoring settings you must plug the drive into one of the USB ports behind the right side of the QuantStudio 12K Flex Instrument touchscreen IMPORTANT Do not use the USB ports on the rear panel of the QuantStudio 12K Flex Instrument The rear USB ports are for use by Life Technologies personnel only to service the instrument Note The backup feature can be used as an administrative tool to manage QuantStudio 12K Flex Instruments You can use the feature to create a standard image for a QuantStudio 12K Flex Instrument that can then be restored on other instruments to bypass the manual setup process Backing up the QuantStudio 12K Flex Instrument settings 1 Plug a USB drive into the USB port on the right side of the QuantStudio 12K Flex Instrument touchscreen 2 If the instrument is in standby touch the QuantStudio 12K Flex Instrument touchscreen to activate it then touch G 3 In the Main Menu touch Tools then touch B Back Up 4 In the Backup Settings screen touch Backup 5 Touch KM to return to the Main Menu 6 Unplug the USB drive Note For administrative purposes you can reuse the instrument settings saved to the USB drive to configure more than one QuantStudio 12K Flex Instruments Note that you must configure the network settings for each instrument individually Restoring the instrument settings
88. verification consumable appropriate for your instrument m Preparing the TaqMan RNase P Instrument Verification Plate 65 E Preparing an array card for instrument verification lt 0 eee 66 Required materials e Centrifuge with plate adapter e Powder free gloves e Safety goggles s TaqMan RNase P Fast 96 Well Instrument Verification Plate Prepare the TaqMan RNase P plate 1 Obtain the TaqMan RNase P Instrument Verification Plate from the freezer then allow the plate to warm to room temperature for approximately 5 minutes IMPORTANT Do not remove the plate from its packaging until you are ready to run it The fluorescent dyes in the dye plate are photosensitive Prolonged exposure to light can diminish the fluorescence signal strength of the plate 2 Remove the RNase P plate from its packaging Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 65 Calibrating Multi Well Plate and Array Card Sample Blocks Verifying the instrument performance 3 Briefly vortex and centrifuge the RNase P plate a Vortex the plate for 5 seconds b Centrifuge the reaction plate for 2 minutes at less than 1500 rpm IMPORTANT The reaction plate must be well mixed and centrifuged c Verify that the liquid is at the bottom of each well of the reaction plate If not centrifuge the reaction plate again at a greater rpm and for a longer time IMPORTANT Do not
89. well separated by tab characters If a well contains more than one assay target the QuantStudio 12K Flex Software lists the data for each additional assay on separate rows repeating the well number and sample information This section describes the following results data formats E Standard curve relative standard curve and comparative CT 216 R Biological replicate resulta 6 6 eee eens 217 W Technical replicate results sai mei a a d eee eens 218 B Genotyping issn deta de bead sane dbadd a ai oiia a EA d bag siete 219 R Melting curve r aE Tada T 8 PAM Sides site Ah gee ete 220 BHI RM 3s oes eg Seta ete Sa eal ee eee anaes 221 W Presence absence no ndee tices we eho Welch ee one de Wa eset Riana lene oe a ote tghe cote 222 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 215 File Format Reference QuantStudio12KFlex export format Standard curve relative standard curve and comparative Cry The following table describes the results data exported from standard curve relative standard curve and comparative Cy experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 character string Target Name Th
90. 00 STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 Unknown B 10000 A B C D E F G H The figure to the right illustrates the arrangement of the standard and unknown populations on a 384 well RNase P plate The RNase P plate contains five replicate groups of standards 1250 2500 5000 10000 and 20000 copies two unknown populations 5 000 and 10 000 copies and a no template control NTC Unknown A Unknown B 5000 10000 Standard 1250 9 Standard 2500 b da Standard 5000 Standard 20000 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 63 Calibrating Multi Well Plate and Array Card Sample Blocks Verifying the instrument performance About the analysis Array Card RNase P Kits The RNase P Kits include one empty array card and eight tubes of solution Each tube contains reaction mix TaqMan Universal PCR Master Mix RNase P primers and FAM MGB dye labeled probe and a known concentration of human genomic DNA template To perform an instrument verification run each solution is loaded into the empty array card in the arrangement shown right When complete the array card contains five replicate groups of standards 200 400 800 1600 and 3200 copies two of unknown populations 800 and 1600 copies and one that serves as a no template control NTC
91. 0D 200 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix E File Format Reference Plate setup file format Plate setup file format You can use plate setup files to automatically populate setup information into an open experiment in the QuantStudio 12K Flex Software or into new experiments created by the command line application see page 189 A plate setup file is a tab delimited ASCII text file txt that contains data that describes the location experiment data information The files can be created manually using a text processor or generated automatically by third party applications IMPORTANT To guarantee successful import of the plate setup file into a experiment the file must contain all the elements described in the following section and in the order that they appear File structure The plate setup file consists of a header which specifies the instrument model for which the experiment is designed and a sample setup section Section Description See Plate setup file header Defines the instrument model for which the page 201 experiment is designed and the dye used as the passive reference Plate setup file body Defines the contents of a 96 384 well plate or array page 202 card including target SNP assay sample and task assignments Plate setup file The plate setup file begins with a header that consists of two lines Each line
92. 2 Also called variant homozygote e Heterozygous samples having both allele 1 and allele 2 Samples having both allele 1 and allele 2 See also genotyping experiment In the thermal profile the stage that holds the temperature constant for a defined period of time A stage that includes one or more steps You can add a holding stage to the thermal profile to activate enzymes to inactivate enzymes or to incubate a reaction Samples having only allele 1 or only allele 2 See also genotyping experiment A gene that is involved in basic cellular functions and that may be constitutively expressed Housekeeping genes may be candidates for use as endogenous controls however their constancy should always be validated experimentally See also endogenous control A software feature that allows you to import plate setup information or security settings before an experiment run You can also import information into some libraries in the system A software feature that allows you to view information about instruments on the network In the Instrument Console you can monitor the status of any instrument on the network view calibration maintenance and instrument properties for a selected instrument and open and close the instrument drawer A software feature that allows you to view information about instrument available on the network In the Instrument Manager you can monitor the status of an instrument monitor amplification plots a
93. 2 Go to Performing the calibration on page 56 Before using each dye plate vortex the plate for 5 seconds centrifuge it for 2 minutes at less than 1500 rpm then confirm that the liquid in each dye plate is at the bottom of the wells If not centrifuge the plate again at a higher rpm and for a longer period of time Correct Incorrect Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time IMPORTANT The dye plates must be well mixed and centrifuged Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 55 Calibrating Multi Well Plate and Array Card Sample Blocks Dye calibration Performing the IMPORTANT The QuantStudio 12K Flex Software guides you through the calibration calibration of each dye separately You must set up run and analyze each dye independently 1 In the Home screen of the QuantStudio 12K Flex Software click Instrument Console 2 In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it 3 After the QuantStudio 12K Flex Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenan
94. 2 user name restrictions 139 user report 145 user role 143 security auditing and electronic signature module See audit See security security users account suspension 154 login 154 overview 154 password change 154 permissions 154 session timeout 155 self test performing 165 serial factor 265 serial numbers 98 serial port 25 28 service pack updates 115 session timeout 139 155 set up instrument security 172 software security 139 settings date time 168 instrument name 168 instrument security 172 maintenance reminders 169 network instrument 170 system shortcuts 171 setup and load plates 98 setup file 191 signing 155 signing electronic signature 155 slope 265 Smart Monitoring 170 Index SMTP requirement 134 SNP 265 SNP assay 265 SNP Assay Library 265 software instrument 30 licenses maintenance 117 robot 31 third party 31 software update 116 specification installation 65 95 specifications installation 65 95 instrument 20 stage 265 staker sealer 37 236 standard 265 standard curve 266 standard curve method 266 standard quantity 266 standards EMC 244 safety 244 standby mode 176 standby time out 168 starting quantity 266 static IP support 126 statistics instrument 171 step 266 storage instrument 178 surge protector requirements 29 SYBR Green dye 266 symbols safety 238 system configuration history contents 147 display 146 149 152 system dye 267 system dyes 19 53 91 System Sel
95. 22 environmental requirements 22 Ethernet port 126 exhaust venting 21 filter sets 19 fuse replacement 114 heated cover temperature 168 icon 168 installation 180 installation specification 65 95 IPV4LL 126 layout and connections 23 log 173 maintenance 36 82 163 maintenance reminders 169 mDNS DNS support 126 moving 179 name setting 168 network setting 170 networking 126 129 272 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide instrument normalization calibration 59 operation safety 241 power on off 176 177 RNase P experiment 63 95 ROI calibration 41 security 172 self test 165 settings 110 164 Smart Monitoring 170 software 30 specifications 20 standby 176 standby time out 168 static IP support 126 statistics 171 storage 178 system shortcuts 171 touchscreen 24 158 uniformity calibration 49 87 verification 37 65 66 96 instrument adapter software 31 Instrument Console 258 instrument control program ICP 31 Instrument Manager 258 instrument verification perform 68 103 troubleshoot 77 105 internal positive control IPC 259 inventoried assays 259 IP settings Ethernet port 170 IPC blocking agent 259 IPv4 link local IPV4LL 126 is signed field 155 K keys software 117 L laser classification 243 laser safety bar code scanner 243 requirements 243 layout instrument 23 network 127 LED 24 Index License Central 117
96. 262 recommended maintenance schedule 36 82 reference 262 reference sample 263 refSNP ID 263 region of interest ROI calibration 263 registration software 117 regression coefficient 262 263 regression line 263 regulator power line 29 reinstalling the instrument 180 reject well 263 relative standard curve method 263 reminders calibration 134 remote monitoring computer setup 130 guidelines 132 instrument 132 instrument setup 129 removal outlier 68 repetitive motion safety 243 replace instrument fuses 114 replicate group 263 replicates 263 reporter 264 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 275 Index reports action log 146 148 149 152 audit 148 electronic signature 152 object audit history 146 149 152 system configuration history 146 147 149 152 user 145 requirements component clearances and positioning 21 computer 30 electrical 22 environmental 22 exhaust venting 21 physical clearances 21 SMTP server 134 weight 20 restore audit records 148 instrument settings 164 results transfer to USB drive 162 reverse primer 264 reverse transcriptase 264 RNase P instrument verification experiment 63 95 kits 63 95 outlier removal 68 perform 63 95 preparation 65 96 R2 value 68 troubleshoot 77 105 when to perform 63 95 robot 27 components 27 28 racks 28 software 31 ROI calibration 41 data 41 perform 43 preparation 42 troubleshoot 72 w
97. 2K Flex Instrument for the associated bin at the designated cycle Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom HRM Raw The following table describes the raw data exported from high resolution melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1 96 384 t Reading 1 based index of the reading Integer Temperature Temperature in Celsius Float Fluorescence Fluorescence value Float Derivative Value of the fluorescence curve derivative for this Float reading point Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 213 E File Format Reference QuantStudio12KFlex export format Amplification data The QuantStudio 12K Flex Software can export the processed amplification data used to generate the amplification plot of a real time PCR experiment The amplification data R are the raw fluorescence readings collected by the QuantStudio 12K Flex Instrument normalized to the fluorescence from the passive reference If available the exported amplification data also exports the baseline compensated normaliz
98. 4 YZ Table 4 Troubleshooting uniformity calibrations Problem symptom Possible cause Action Uniformity calibration failed Abnormally low spectra across all wells of the plate or array card Confirm that you loaded an ROI plate or array card into the QuantStudio 12K Flex Instrument If not perform the calibration again using the correct ROI plate or array card If you are using the correct plate or array card perform the calibration again using a different ROI plate or array card If the calibration fails again contact Life Technologies technical support One or more wells produced spectra that deviate significantly from the rest of the plate or array card While viewing the calibration data in the Analysis screen locate the wells with abnormal signal in the Plate Layout tab Rotate the calibration plate or array card 180 then perform the calibration again Determine the location of the contaminated wells again If the position s of the well s identified in step 1 and step 2 are e Identical The sample block is contaminated Decontaminate the sample block e Reversed The ROI plate or array card is contaminated Discard the plate or array card then perform the uniformity calibration using a new ROI plate or array card If the calibration fails again contact Life Technologies technical support Instrument does not eject the ROI plate The adhesive cover may ha
99. 6 909 u 0 SZ 86 98u u 0 SZ 86 98u ZOTOD 4 612L TH TH THD THD THD THD THD UvA awMoo CERIS AWAD AVAD UWAOO AWAD wey 61eL ZoToD dnozrbotg u SZ 0 0 999 u S700 duu u S700 58u u 0 SZ 01999 u 0 GZ 01999 u 0 GZ 01999 u 00 SZ 999 u GZ 0701999 u 0 GZ 01999 u 0 SZ 01999 u 0 SZ 01999 u 0 0 SZ 999 u 0 0 GZ 98u u 0 0 GZ 98u Sure dnozbotg ZoTOD eTdues xou sajdurexy uau ad xa sty ul pasn jou ore sdnois pestsojorg GOW OxAN teyouenb yuessaronyj uou y pue p Jayodar INVA 94 YIM paid et are saqoid y shesse gueybey yoq 104 uresq pue JIeay BATT ay WOJ VN s jdwes aarut ul LHD pue TY 131 981e om Jo uorssaidxa ay sayenyead yusuttiadxea ay wiayshS X AZI ne OEPMS END e UO UNI aq 0 yUaIUTIAadxa Yd eaayeyuenb e 101 pazeard apy dny s ayeyd e smoys jdurex SuLMoT O ay sjUuaWadxa Yq aaeyUenH YMO O YMO O VNO 2 UNa UNa UNa UNa UNa YNO2 YNO2 YNO2 YMO O Sue EEIT EEIT EES EES EEIT EEIT I A TI I A TI I A TI I A TI I A TI I AFT etdues HANNS NN Y O TT M dnjes etdues eouezeyey eATSsseg x odkl quownazqsur x UNa WNdO YMO O WNdO WNdo UNa UNa UNa UNa UNa YNO2 YNO2 WNG YNO O Sue u eag u eag u eag EEIT EEIT EEIT TeAYT utezg EEIT qreeH EEIT TeAYT TeAYT TeAYT etdues PBX E8E Z8E T8E 08E GLE 8LE A r T TT M dnjes at dues
100. 9 0 6 kg 171 5 0 13 Ibs Verify that the path to transport the QuantStudio 12K Flex Instrument is clear of obstructions Enlist at least one other person to lift and carry the QuantStudio 12K Flex Instrument Keep your spine in a good neutral position Bend at the knees and lift with your legs Do not lift an object and twist your torso at the same time Coordinate your intentions with your assistant before lifting and carrying Reconnect the components of the QuantStudio 12K Flex System Use the Ethernet cable supplied with the QuantStudio 12K Flex System to connect the QuantStudio 12K Flex Instrument Ethernet S Ethernet port to the network interface card in the computer IMPORTANT Do not use a standard Ethernet cable to connect the QuantStudio 12K Flex Instrument to the computer en IMPORTANT Do not connect the Ethernet cable to the Ethernet 2 port on the QuantStudio 12K Flex Instrument The second port is for Life Technologies service use only Install the sample block and heated cover assemblies Perform a RNase P instrument verification run If the run Passes Do not recalibrate the QuantStudio 12K Flex System No further action is necessary Fails Perform the following calibrations in the specified order ROI background uniformity dye then normalization calibrations 180 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance
101. ANDARD IPC NTC or BlockedIPC Reporter The reporter dye that labels the probe for the 100 character string target assay Quencher The quencher dye that labels the probe for the 100 character string target assay Quantity Standard quantity if applicable This column Float or Integer only appears for Standard Curve and Relative Standard Curve experiments Comments Additional text that describes the well 1024 character string Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Contains rJed b lue and g reen color values each between 0 255 The field is enclosed in double quotes with no spaces between the values Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 211 212 File Format Reference QuantStudio12KFlex export format Genotyping experiments The table below describes the sample setup data that can be exported from a genotyping experiment The body can contain all or some of the data columns below depending on the export configuration Note For all other experiments see Quantification and presence absence experiments on page 211 Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 character string Sample Color The RGB color of
102. ARD During edge of the OpenArray plate case lid is askew instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature QC image shows misalignment of the OpenArray plate see below 1 Unload the OpenArray plate from the QuantStudio 12K Flex Instrument 2 Hold the OpenArray plate up to a light source and verify that the lid is positioned correctly If the lid is not seated correctly on the case then the through holes along the unsealed edge of the OpenArray plate will fail to amplify 3 Order and perform a replacement RNase P experiment If the replacement OpenArray plate fails contact Life Technologies for further assistance 108 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Maintenance This chapter covers S Regular data maintenance ene 110 S Decontaminating the sample block 6 06 o cece eens 111 m Replacing the instrument fuses e eee nes 114 E Updating the Windows operating system 6 6 c cece 115 S Updating the QuantStudio 12K Flex Software and Firmware 116 m Managing QuantStudio 12K Flex Software licenses asusunu 117 S Replacing the sample block 119 S Replacing the heated Over 6c cee eens 121 R Replacing the plate adapter 0 cece
103. Allele2 Name The name of the second allele detected by the SNP assay lt 100 character string gt Allele2 Color The RGB color used to represent data for the second allele RGB lt r gt lt g gt lt b gt nB Allele2 Reporter The reporter dye used to label the probe for the second allele lt dye name gt 88 Allele2 Quencher The quencher dye used to label the probe for the second allele lt dye name gt 88 T Cannot be blank Contains red blue and g reen color values between 0 255 The field must be set within double quotes with no spaces between the values Can be empty if the Task field is empty Otherwise the field must contain a value ttSee the Applied Biosystems QuantStudio 12K Flex Real Time PCR System Getting Started Guide to determine the tasks applicable to your experiment The dye must already exist in the QuantStudio 12K Flex Software Dye Library The dye name must be 100 characters or less Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 203 qOW OAN aOW OAN 9W AN 9W AN 9W AN 9W AN 9W AN 9W AN aOW OAN aOW OAN aOW OAN a OW O4N squewuog AqtTquend zAseyouend WW WW zeqroday NMONANN SNONASOCNaA NMON MNOA SNONASOGNa NMONANN SNONASOGNa NMONMNQO SNONASOGNa NMON MNQO SNONJ JOANA NMONANN SNONASOGNaA KI u SOT 0 0 999 0 SZ 86 duu SOT 0 0 duu z s6 duu SOT
104. Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 103 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance 8 In the Amplification Plot select Cnr vs Well from the Plot Type menu then verify the uniformity of the Cy values for the replicate population a In the plate layout select all through holes b In the plot verify that the Crs of the replicate population are equivalent c If an outlier is present in the population select the corresponding through hole of the plate layout then click Omit to remove the through hole from the analysis If the total number of outliers for the replicate population exceeds 48 through holes repeat the experiment using another OpenArray plate 9 Review the Results Table for quality flags generated by the experiment a Select the Results Table tab b Review the Flag column for through holes that generated quality flags c Troubleshoot each through hole that generated a flag as explained in Troubleshooting on page 105 AMPNC Amplification in negative control BADROx Bad passive reference signal BLFAIL Baseline algorithm failed CTFAIL Cy algorithm failed EXPFAIL Exponential algorithm failed HIGHSD High standard deviation in replicate group NOAMP No amplification NOISE Noise higher than others in plate NOSIGNAL No signal in through hole OFFSCALE Fluorescence is offscale OUTLIE
105. Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 61 Normalization calibration Calibrating Multi Well Plate and Array Card Sample Blocks IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the plate remove the plate according to Table 6 Troubleshooting normalization calibrations on page 76 9 Discard or store the plate or array card Consumable Action Array card Discard the array card Plate Return the normalization calibration plate to its packaging sleeve then return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate is stored in its original packaging sleeve you can use it to calibrate a QuantStudio 12K Flex System 3 times for up to 6 months after you open it 10 In the Normalization Calibration screen click Finish to complete the calibration then click Yes when prompted to save the results 11 Repeat steps 4 through 10 to perform the remaining normalization calibration 62 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Verifying the instrument performance Verifying the instrument performance When to perform the RNase P experiment About the RNase P kits Perform the RNase P instrument verification experiment to verify the performance of
106. At the bottom of the wells centrifuge the plate for a longer time then repeat the calibration e Equivalent in volume the plate is not sealed and the reagents have evaporated Discard the plate and run another 2 If the dye calibration plate appears to be normal discard the plate and run another 3 If the problem persists contact Life Technologies If you are running a custom dye calibration plate create another plate but increase the concentration of the dye that produced insufficient signal e Spectra contain peaks in more than one filters e One or more raw spectra exceed the maximum limit for the QuantStudio 12K Flex System Fluorescent contaminants are present on the sample block or dye calibration plate If you are running a custom spectral calibration plate the dye may be too concentrated Verify that contaminants are not present by performing a background calibration see Background calibration on page 45 If the background calibration does not show sample block contamination the dye calibration plate may be contaminated Note If you are running a custom dye calibration plate create another plate but decrease the concentration of the dye that exceeded the detectable limit Instrument does not eject the dye plate The adhesive cover may have adhered the plate to the heated cover within the instrument 1 Power off the QuantStudio 12K Flex Instrument 2 Wait for 15 minutes then po
107. Change the Status from ACTIVE to INACTIVE then click Save 142 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Determining the name of the logged in user Create or edit a user role Chapter 6 Security Audit and Electronic Signature 6 Managing user accounts TM The title bar of the QuantStudio 12K Flex Software window displays the name of the user User roles determine the permissions associated with a user account The QuantStudio 12K Flex Software includes three default user roles e Administrator cannot be edited or deleted e Scientist e Technician You can modify the Scientist and Technician roles and you can create your own roles with customized settings as needed To determine the permissions for a default role or to edit it select the role then click Edit Creating a user role 1 In the QuantStudio 12K Flex Software select Tools gt Security gt Settings 2 In the Security Settings dialog box select the Roles tab 3 Click Create 4 Enter a role name and optional description 5 Select permissions see Permissions and default user roles on page 144 To select all permissions in a category select the check box next to the category Note Operations not shown in the following table are available to all user roles 6 Click Save Role Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Adminis
108. Defines the ID of the Assay Plate Normally this 100 character string is a barcode printed on the plate XXX Setup File Version lt version number gt Output Plate Size lt number of wells gt Output Plate ID lt plate id gt Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 223 E File Format Reference 7900 export format Assay detector data Well data The assay data describes the qualities of the target assays present on the consumable In the context of the 7900HT System target assays are referred to as detectors The section consists of multiple lines that define the total target assays followed by a column header and tab separated data The first line defines the total number of target assays on the consumable formatted as follows XXX Number of Detectors lt number of assays gt The column header defines the columns of exported data followed by one or more lines where each row defines the properties of a single assay separated by tab characters Column Description Output Detector The name of one target in the well if applicable 100 character string If a well contains multiple targets one row is used per target Reporter The reporter dye that labels the probe for the 100 character string target assay Quencher The quencher dye that labels the probe for the 100 character string target assay Description The stand
109. Guide Appendix A Manual Instrument Operation Administering the instrument from the touchscreen Viewing the You can use the QuantStudio 12K Flex Instrument touchscreen to view a log that instrument log summarizes instrument activity from the last 6 months For each recorded activity the activity log provides a description of the activity and the date time when it occurred Viewing the log 1 If the instrument is in standby touch the QuantStudio 12K Flex System touchscreen to activate it then press 4 2 In the Main Menu touch Tools then touch View Log 3 In the View Log screen configure the settings to display the records of interest e Select an option from the drop down menu to filter the log e Select Earliest First or Latest First to determine the order to sort the records 4 Touch Q to return to the Main Menu Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 173 A Manual Instrument Operation Administering the instrument from the touchscreen 174 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Powering On or Off Storing and Moving the System This appendix covers Placing the QuantStudio 12K Flex System on standby 5 176 Powering on the QuantStudio 12K Flex System 6 cece ee eee 176 Powering off the QuantStudio 12K Flex System 0 ccc e ee
110. IX A Manual Instrument Operation 05 157 Instrument touchscreen functions cee nanen ranere rnrn nnn 158 List of instrument functions 0 cece een eee ee nett eens 158 Operating the instrument from the touchscreen cece cette eeeeee 159 Creating an experiment from a template 02 cc cece eee 159 RUNING an experiMment cnis 22s oe heat Seb phe ee eh pee eee ee 160 Transferring experiments templates and results data eee eee 161 Maintaining the instrument from the touchscreen 0200 cece cece eee e eens 163 Backing up and restoring the instrument settings n cece e eee 164 Performing an instrument self test 02002 c ccc eee eee eae 165 Updating the instrument firmware 0600 cee eee eee teen eee aes 166 Administering the instrument from the touchscreen 0000c eee e eee e eee eee ees 167 Defining the date and time 00 cece eee eee enna 168 Defining the instrument settings 0002 c eect eee eee 168 Defining the maintenance reminders cece eee eee nena 169 Defining the network settingS ccc cece rnrn nnr rnrn rrna 170 Defining the system shortcuts cece cece eee eee eee aes 171 Reviewing the instrument statistics 00000 cee eee eee eens 171 Enabling disabling instrument security 0 00 e cece cece nets 172 Viewing the instrument log 0c e eee eee e eee eee eeees 173 APPENDIX B Powering On or Off Storing and Moving the Syst
111. In the Main Menu touch Browse Experiments In the Browse Experiments screen select the experiment a Touch m then touch USB b Touch the desired experiment then touch 6 Save In the Save Experiment As screen touch the experiment that you want to transfer to the USB drive then touch Save Touch Q to return to the Main Menu Unplug the USB drive Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 161 A Manual Instrument Operation Operating the instrument from the touchscreen Copying experiment results to a USB drive 1 Plug a USB drive into the USB port on the right side of the touchscreen 2 If the instrument is in standby touch the QuantStudio 12K Flex Instrument touchscreen to activate it then press 0 3 In the Main Menu touch Collect Results 4 In the list of experiments touch the row s for the experiment s of interest or touch E Select All 5 Touch g Copy to USB 6 Inthe Copy Results To USB screen check that the name of the USB drive is correct to ensure that it is mounted then touch Z Copy to USB 7 Touch a to return to the Main Menu 8 Unplug the USB drive Note After the results from a completed run have been collected the corresponding experiment displays Collected and it can be deleted 162 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix A Manual Instr
112. K Flex Instrument using a USB flash drive Before transferring data you must plug the drive into one of the USB ports behind the right side of the QuantStudio 12K Flex Instrument touchscreen templates and results data IMPORTANT Do not use the USB ports on the rear panel of the QuantStudio 12K Flex Instrument The rear USB ports are for use by Life Technologies personnel only Appendix A Manual Instrument Operation Operating the instrument from the touchscreen Transferring templates from a USB drive 1 2 Plug a USB drive into the USB port on the right side of the touchscreen If the instrument is in standby touch the QuantStudio 12K Flex Instrument touchscreen to activate it then press G In the Main Menu touch View Templates In the Browse Experiments screen select the template a Touch e then touch USB b Touch the desired template then touch Save In the Save Experiment As screen set the name for the file a Touch the New Template Name field then enter a name for the copied file b Touch the Save to Folder field then select the folder to receive the file c Touch Save Touch Q to return to the Main Menu Unplug the USB drive Transferring experiments from a USB drive 1 2 Plug a USB drive into the USB port on the right side of the touchscreen If the instrument is in standby touch the QuantStudio 12K Flex Instrument touchscreen to activate it then press G
113. MPORTANT A user must be present throughout the duration of the calibration Following the first read the OpenArray Calibration Plaque must be rotated 180 before the instrument can complete the calibration 84 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Load the plaque 1 Chapter 3 Calibrating OpenArray Plate Sample Blocks Background calibration When the instrument door opens load the OpenArray Calibration Plaque black side up into the plate retainer IMPORTANT Ensure that the OpenArray Calibration Plaque is loaded into the plate retainer so that the black side of the tool is facing up IMPORTANT The instrument should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained Start the calibration a Select Check the box when the calibration plaque has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature Rotate the plaque When the instrument door opens and you are prompted to rotate the OpenArray Calibration Plaque 1 Rotate the OpenArray Calibration Plaque 180 then place
114. MRA Dye 4432296 384 Well Region of Interest ROI and Background Plates 4432320 e 384 Well Region of Interest ROI Calibration Plate e 384 Well Background Plate 384 Well Normalization Plates with FAM ROX and VIC ROX 4432308 Dyes e 384 Well Normalization Plate with FAM ROX Dye e 384 Well Normalization Plate with VIC ROX Dye Kit TaqMan RNase P Fast 384 Well Instrument Verification Plate 4455280 s 384 Well TaqMan RNase P Fast Instrument Verification Plate Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 233 Parts and Materials Calibration and verification kits 96 well sample block kits QuantStudio 12K Flex System consumable Part no S 96 Well Spectral Calibration Plate with FAM Dye 4432327 15 to 25 96 Well Spectral Calibration Plate with VIC Dye 4432334 96 Well Spectral Calibration Plate with ROX Dye 4432340 96 Well Spectral Calibration Plate with SYBR Green Dye 4432346 96 Well Spectral Calibration Plate with TAMRA Dye 4432352 96 Well Spectral Calibration Plate with NED Dye 4432358 96 Well Region of Interest ROI and Background Plates 4432364 e 96 Well Region of Interest ROI Calibration Plate e 96 Well Background Plate 96 Well Normalization Plates with FAM ROX and VIC ROX 4432370 Dyes e 96 Well Normalization Plate with FAM ROX Dye e 96 Well Norm
115. Main Menu Defining the You can use the QuantStudio 12K Flex Instrument touchscreen screen to maintenance e Set the expiration period for the instrument calibrations and LED replacement reminders e Activate deactivate or change the frequency of the maintenance reminders displayed by the QuantStudio 12K Flex Instrument Setting the reminders 1 Ifthe instrument is in standby touch the QuantStudio 12K Flex System touchscreen to activate it then press g 2 In the Main Menu touch Settings then touch Set Maintenance Reminders 3 Configure the maintenance reminders For each maintenance reminder a Touch the Calibration expires after field enter the number of days or hours that should elapse until the association calibration expires then touch Done b Touch the check box to activate or deactivate reminders for the associated calibration c Touch the Display reminders before field enter the number of days before the associated calibration expires that the QuantStudio 12K Flex Instrument should start displaying warnings of the impending expiration then touch Done 4 Touch Save to save the settings then touch OK when prompted 5 Touch Q to return to the Main Menu Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 169 A Manual Instrument Operation Administering the instrument from the touchscreen Defining the 1 If the instrument is in standby tou
116. Maintenance and Administration User Guide Calibrating Multi Well Plate and Array Card Sample Blocks This chapter covers E Recommended calibration and maintenance 36 E Preparing array cards for instrument calibration lt e rnrn 37 ROMcalibrab Onis 2 s 4 4 44 Stes ae he a oe Dh le AA ele eb ead 41 S Background calibration 0 eens 45 R Uniformity calibration se e eens 49 M Dye calibration rRe K N wire Celok reece EE gs ia Woodie ga Wie Meee ee E ele aE 53 E Normalization calibration 0 ccc eet ene ees 59 m Verifying the instrument performance e c cece eee eee 63 L Troubleshooting si sais soa steve vee ein BAe ohne Ge E E apes E EE A EAA 71 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 35 Recommended calibration and maintenance Calibrating Multi Well Plate and Array Card Sample Blocks Recommended calibration and maintenance The Applied Biosystems QuantStudio 12K Flex Real Time PCR System requires regular calibration and maintenance for proper operation The following table displays the recommended maintenance schedule that you must perform to ensure optimal instrument performance IMPORTANT Calibrate the QuantStudio 12K Flex System at the same ambient temperature at which you will run experiments Extreme variations in ambient temperature can affect the heating and cooling of the QuantStudio 12K Flex System and in
117. MicroAmp 96 Well Tray Retainer Set e MicroAmp Optical Adhesive 50 uL Film s MicroAmp Optical 96 Well Reaction Plate with Bar Code 96 well plate e MicroAmp Optical Adhesive 50 uL 0 1 mL Film s MicroAmp Optical 96 Well Fast Reaction Plate with Bar Code 384 well Notched corner e MicroAmp Optical Adhesive 20 uL plate Film e MicroAmp Optical 384 Well Reaction Plate with Bar Code Array card Array card T UL OpenArray OpenArray plate 33 nL plate 32 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 1 Getting Started 1 QuantStudio 12K Flex System consumables Guidelines for Observe the following guidelines when using tubes plates or array cards handling consumables Store the calibration plates or array cards in a dark place until you are ready to use them The fluorescent dyes in the wells of calibration consumables are photosensitive Prolonged exposure to light can diminish the fluorescence of the dyes Do not allow the bottoms of tubes or plates to become dirty Fluids and other contaminants that adhere to the bottoms of the consumables can contaminate the sample block and cause an abnormally high background signal Confirm that the centrifuge you use is clean Before centrifugation wipe down the bucket using a tissue Plates only Vortex all calibration plates to ensure complete mixing then centrifuge them to ensure that al
118. NKNOWN Or NTC RQ The relative quantity calculated for the replicate Float wells of the target sample combination RQ Min The minimum relative quantity calculated for the Float replicate wells of the target sample combination The lower limit of the confidence interval RQ Max The maximum relative quantity calculated for the Float replicate wells of the target sample combination The upper limit of the confidence interval Ct Mean The average C of the replicate wells for the Float specified target sample combination Delta Ct Mean The average AC of the replicate wells for the Float specified target sample combination Delta Ct SD The standard deviation of the AC for the replicate Float well Depending on the analysis settings this column may by replaced with Delta Ct SE the standard error of the AC Delta Delta Ct The AAC value of the replicate wells for the Float specified target sample combination 218 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Genotyping Appendix E File Format Reference QuantStudio12KFlex export format The following table describes the results data exported from genotyping experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1
119. ORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 6 After loading the plate or array card start the calibration a In the Setup tab select Check the box when the background calibration plate has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 47 Calibrating Multi Well Plate and Array Card Sample Blocks Background calibration 7 When the run is complete and the QuantStudio 12K Flex Software displays the Analysis screen confirm the analysis status of the calibration then select the QC tab and review the quality check summary e Analysis Status Indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or the data it collected is unusable Note Abnormal spectra or abnormally high background fluorescence can indicate the presence of contamination on the plate array card or sample block which can cause the calibration to fa
120. Operating the instrument from the touchscreen 0 eee eee eee 159 Maintaining the instrument from the touchscreen 00000 eee eee 163 Administering the instrument from the touchscreen n nssnsnn snn 167 Note This appendix describes how to operate the QuantStudio 12K Flex Instrument manually using the touchscreen interface Although the QuantStudio 12K Flex Instrument can be used without a physical attachment to a computer the touchscreen allows you to perform only a subset of the total instrument functions Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 157 A Manual Instrument Operation Instrument touchscreen functions Instrument touchscreen functions List of instrument functions The QuantStudio 12K Flex Instrument features a touchscreen interface that you can use to run experiments manage instrument settings and configure the QuantStudio 12K Flex Instrument for network use The touchscreen does not provide access to all instrument functions Features such as experiment analysis instrument calibration and remote notification are available only through the QuantStudio 12K Flex Software The following table summarizes the functions that are available from the QuantStudio 12K Flex Instrument touchscreen The table organizes the functions by user role where operational functions are for users that perform experiments maintena
121. PORTANT Perform the following procedure only if you are calibrating a QuantStudio 12K Flex System with an OpenArray plate sample block During a dye calibration the Applied Biosystems QuantStudio 12K Flex Real Time PCR System e Collects spectral data from the FAM dye standard e Stores the spectral information for the dye standard in a dye calibration file The QuantStudio 12K Flex Software uses the pure spectra data during experiment runs to characterize and distinguish the individual contribution of dyes in the total fluorescence collected by the QuantStudio 12K Flex Instrument After each run the QuantStudio 12K Flex Software receives data in the form of a raw spectra signal for each reading It determines the contribution of each fluorescent dye used in the sample by comparing the raw spectra to the pure spectra calibration data When you save an experiment after analysis the QuantStudio 12K Flex Software stores the pure spectra with the collected fluorescence data for that experiment IMPORTANT Calibrate only those dyes that are present in the chemistries that you intend to run on your QuantStudio 12K Flex System e QuantStudio 12K Flex System Installation Calibration Kit OpenArray FAM Dye Solution QuantStudio 12K Flex System OpenArray Calibration Cases 4 QuantStudio 12K Flex System OpenArray Plugs 4 QuantStudio 12K Flex System OpenArray Calibration Syringe a
122. R System Maintenance and Administration User Guide Calibrating OpenArray Plate Sample Blocks This chapter covers E Recommended calibration and maintenance 82 S About the OpenArray Calibration Plaque 0 0 cece eee 83 R Background calibration se c ccc eee eens 84 R Uniformity calibration 0 eee nes 87 E Dye calibration ic 3 ise enta eke Giese hs ete hee OE eh pol ae Bo Gee 90 m Verifying the instrument performance e c cece eee eee 95 R Troubleshooting cos ek ee Les tb ee one Pea 105 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 81 Recommended calibration and maintenance Calibrating OpenArray Plate Sample Blocks Recommended calibration and maintenance The Applied Biosystems QuantStudio 12K Flex Real Time PCR System requires regular calibration and maintenance for proper operation The following table displays the recommended maintenance schedule that you must perform to ensure optimal instrument performance IMPORTANT Calibrate the QuantStudio 12K Flex System at the same ambient temperature at which you will run experiments Extreme variations in ambient temperature can affect the heating and cooling of the QuantStudio 12K Flex System and in extreme cases influence experimental results IMPORTANT Do not use organic solvents to clean the QuantStudio 12K Flex System Table 8 OpenArray plate samp
123. RRG Outlier in replicate group SPIKE Noise spikes THOLDFAIL Thresholding algorithm failed 10 If you omitted outliers click Reanalyze to analyze the run If the status of the RNase P Run is Failed after performing steps 8 through 10 repeat the RNase P experiment using a different RNase P plate If the problem persists contact Life Technologies 11 Complete the calibration as instructed When the QuantStudio 12K Flex Instrument ejects the tray arm then discard the OpenArray plate WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the OpenArray plate remove the plate according to Troubleshooting on page 105 12 Click Finish then click Yes when prompted to save the experiment 104 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide olpnysjueng swaj stsoig panddy wl aping Jasf voONessiulWpy pue adueuajulep W IS S YJd aw jeay Xa4 YZ SOL Troubleshooting Problem symptom Possible cause Action More than the maximum number of outliers are present in RNase P data Possible contamination Pipetting inaccuracy Order and perform a replacement RNase P experiment If the replacement RNase P OpenArray plate fails contact Life Technologies for further as
124. Register Now to create an account When you are connected to the Applied Biosystems Store follow the prompts to complete your order Ordering from the Life Technologies To order Procedure Website Assays and reagents Go to www lifetechnologies com Under I Want to Buy select the product of interest and accessories Click Parts and accessories Calibration kits 1 2 Instrument parts 1 Go to www lifetechnologies com quantstudio 2 3 Select the desired components the complete the order as instructed See Consumables on page 236 for a complete list of compatible instrument parts accessories and kits Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 231 Parts and Materials Accessories Accessories The following accessories are to be used with the Applied Biosystems QuantStudio 12K Flex Real Time PCR System QuantStudio 12K Flex System accessories Part no 384 Well Plate Adapter 4457087 384 Well Array Card Heated Cover 4453555 96 Well Heated Cover 4453560 96 Well Plate Adapter 4459845 96 Well Tube Adapter 4462077 Array Card Plate Adapter 4454166 Fast 96 Well Heated Cover 4459838 Fast 96 Well Plate Adapter 4459846 Fast 96 Well Tube Adapter 4462078 OpenArray Heated Cover 4471049 OpenArray Plate Adapter 4454166 QuantStudio 12K Flex System 384 Well S
125. Studio 12K Flex Real Time PCR System Maintenance and Administration User Guide 193 D Command line Software Operation Command syntax and arguments Command syntax and arguments Batch file creation The command used to create batches of files uses the following syntax cmdlineutil ex xpgen parameters The following table lists the acceptable parameters that can be included in any order See Examples on page 197 for an example of the experiment creation command IMPORTANT Enclose file paths in double quotes to allow spaces in the string L lt filepath gt Optional Specifies the path and name lt filepath gt of the assay information file aif or aix that the software uses to create new experiment files Example a D assayfiles assayfile aif lt filepath gt Optional Specifies the path and name lt filepath gt of the barcode file that the software uses to create new files If the b parameter is not used then the software creates the number of experiment specified by the n parameter Example b D barcodefiles barcodefile txt lt string gt Optional When the f parameter is included specifies the alphanumeric string that the software includes in the file names of the new experiments If no value is supplied custom is used as the default value Example c Batch001_ lt option gt Optional Specifies the convention that the software uses to name the new files
126. USER GUIDE applied biosystems by Life technologies Applied Biosystems QuantStudio 12K Flex Real Time PCR System MAINTENANCE AND ADMINISTRATION Publication Part Number 4470689 Rev B Revision Date 6 15 12 6 technologies For Research Use Only Not for human or animal therapeutic or diagnostic use Information in this document is subject to change without notice WARRANTY AND LIABILITY LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PURCHASER Label License No right is conveyed expressly by implication or by estoppel under any other patent claim such as claims to apparatus reagents kits or methods such as 5 nuclease methods other than apparatus claims covering the purchased apparatus Further information on purchasing licenses may be obtained by contacting the Director of Licensing Life Technologies 5791 Van Allen Way Carlsbad CA 92008 USA TRADEMARKS The trad
127. Verifying the instrument performance 3 Using a permanent marker mark the side of the empty array card with RNase P 4 Transfer 100 uL of each solution into the appropriate port of the array card 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 PORT For each transfer a Place the array card on a lab bench with the foil side down b Load 100 uL of fluid into a pipette c Hold the pipette in an angled position 45 and place the tip into the fill port There is a fill port on the left arm of each fill reservoir the larger of the two holes Fill Z port 1 2 8 4 5 6 IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port d Dispense the fluid so that it sweeps in and around the fill reservoir toward the vent port When pipetting the reagents into the array card pipet the entire 100 uL volume into the fill reservoir but do not go past the first stop of pipettor plunger or you may blow the solution out of the port IMPORTANT Do not allow the tip to contact and possibly damage the coated foil below the fill port Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 67 Calibrating Multi Well Plate and Array Card Sample Blocks Veri
128. WN kA Liver cDNA RGB 25 0 0 Liver cDNA RGB Liver cDNA RGB Heart cDNA RGB Heart cDNA RGB 25 0 0 25 0 0 072570 0 25 0 202 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Plate setup data columns Appendix E File Format Reference Plate setup file format The following table lists the headings and columns that are present in the plate setup file body of all experiment types followed by the columns that are specific to genotyping experiments and non genotyping experiments Column name Description Valid values Well The number of the well on the consumable where the well lt Positive integer 1 96 384 gt 4 numbers start at 1 for well A1 upper left corner and increase 0 from left to right and from top to bottom Sample Name The name of the sample contained by the associated well lt 100 character string gt 8 Sample Color Optional The RGB color of the associated sample RGB lt r gt lt g gt lt b gt 8 x Biogroup Name Optional The name of the associated biological group lt 100 character string gt a Biogroup Color Optional The RGB color of the biological group RGB lt r gt lt g gt lt b gt 8 Comments Optional Additional text that describes the well lt 1024 character string gt Target Name The name of the target detected or amplified
129. a cote tee metas on a ite ah eh ce aa ah ee EM gE a OP al ae ect ah hh Oe a Ae 245 General chemical safety 0 000 cece rnnr raara errena 245 SDSS esi ren eect T 246 Chemical waste safety 2 2 c2054 cce4cbled ie ceeehs eed gecths gen SAREE G ee RSS ede 246 Biological hazard safety 248 Safety alerts cat eae felted a La detuned hte te bee a ec Red ltt nas dat 249 General alerts forall chemical 249 General alerts for instrumentation 0 00 cece e eee ene n nents 249 Specific alerts for instrumentation 0 0c c cece eee eee rrna 249 Documentation and Support 00 e e e eee eee eee eens 251 Related documentation lt gka a Z R EL cae eee eee da ede Lae dee A AKE 251 Obtaining information from the Help system 20 cece nnana 252 Obtaining SUPPO heidan a a aa e ak Pp tele ato ar e ects hl 252 Glossary cerea A a a RR 253 WIC Kaas a shit aasa Ea cc A este E EE EE AAA Tag 269 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 11 Contents 12 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Purpose Audience Assumptions About This Guide The Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration Guide provides reference information for the QuantStudio 12K Flex Instrument and describes how to prepare maintain and troubleshoot the system This use
130. a well contains more than one assay target the QuantStudio 12K Flex Software lists the data for each additional assay on separate rows repeating the well number and sample information The data included in the sample setup data export varies depending on experiment type This section describes the following sample setup data formats S Quantification and presence absence experiments 00000 e eee 211 R Genotyping experiments 0 ene eee tenes 212 Quantification and presence absence experiments The table below describes the sample setup data that can be exported from absolute quantification relative quantification or presence absence experiments The body can contain all or some of the data columns below depending on the export configuration Note For genotyping experiments see Genotyping experiments on page 212 Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 character string Sample Color The RGB color of the associated sample RGB lt r gt lt g gt lt b gt 8 Target Name The name of one target in the well if applicable 100 character string If a well contains multiple targets one row is used per target Target Color The RGB color of the associated SNP assay RGB lt r gt lt g gt lt b gt nB Task The task the target is used for in this well UNKNOWN ST
131. acterstring Custom Results table of the experiment per field Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix E File Format Reference QuantStudio12KFlex export format HRM The following table describes the results data exported from high resolution melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 character string Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN Or NTC Reporter The reporter dye that labels the probe for the 100 character string target assay Quencher The quencher dye that labels the probe for the 100 character string target assay Variant Calls The variant call assigned to the sample at the Hetero Homo 1 or specified well Homo 2 Confidence The calculated confidence of the automatic variant Float 1 100 Value call CT The calculated threshold cycle C for the target Float at the specified well Ct Me
132. actions that require signature 0000 cece eee eee 151 How the software prompts electronic signature 20e cece eee nnn 152 Generating electronic signature reports 0c ccc rrn rnrn rrr rnrn rrr 152 Displaying electronic signature records cece eee ences 152 Saving or printing electronic signature records cece eee cece eens 152 Saving or printing the table of electronic signature events 2 2202e eee eee 153 Exporting and importing settingS cect eee eee eae 153 Exporting settingS snurrer ved ppd ovina heed oe oe ted bie tee eet dee cere eee ores 153 MPOrtiNG SettiNGS ves cide ce yee pees edd pe gles sean kent mew RAR X acid 153 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 7 Contents DSEG ante 2 2 a dan Geto a LEA RA ges R tees aaa dle tarde aaa ie PR 154 S GUPILY rare a Pee BP R he HR eed A a dd ahd ne ea Gabe a eed nat T TA caer ees 154 LOGGING UN a his din te eee d Ome Thad She eae et uy ate oe yee tis Mince 154 PErMmiSSIONS S fcr a aa ra Soom R R Mane AR Sans eae c ee Guta a aet 154 Changing your password when it expires ccc cece cee eens 154 Account suspension 222200 a a eee eee eee neces dE a 154 Session tIME OUt iseia een teen eet tee nent N nents 155 AU Cito coasters Wire 0 0 A Abie aed Age ee ald gate A Washed Roma ad TATE A SER he 155 Electronic Signature 2 02 a cant edgar dtnata laine eee eed ote ad 155 APPEND
133. al to generate a set of experiment files cmdlineutil ex xpgen t C Applied Biosystems QuantStudio 12K Flex Software User Files experiments templates standard_curve edt a C Applied Biosystems QuantStudio 12K Flex Software User Files experiments examples AIF AIF_820629 txt s C Applied Biosystems QuantStudio 12K Flex Software User Files experiments examples Plate Setup Files SDS_820629 txt m C Applied Biosystems QuantStudio 12K Flex Software User Files experiments examples SampleNames SampleFileNames txt c alloptionsused f Plate Barcode_Custom Name Field b C barcodes txt v 1 C Experiment For this example the command line application e Imports assay definitions from the AIF_820629 txt assay information file e Imports sample names from the SampleFileNames txt sample file e Generates an experiment for each barcode in the barcodes barcodes txt barcode file where each new experiment uses the settings found in the standard_curve edt template file and the sps_820629 txt setup file Note The setup file links the information from the AIF_820629 txt and SampleFileNames txt to each new experiment file e Saves all generated files using the following naming convention lt barcode gt _alloptionsused e Saves all generated files to C Experiment lt date time gt Note The command line application automatically creates a time stamped folder at the export location for each batch
134. al that can receive or supply alternating current or voltage ILO C GO Indicates that the device receives or supplies direct current or voltage Indicates the On Off position of a push push main power switch uo Indicates a terminal that can receive or supply alternating or direct current or voltage Safety symbols The following table describes the safety symbols that may be displayed on Life Technologies devices Each symbol may appear by itself or with text that explains the relevant hazard These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents Symbol Description ZN Indicates that you should proceed with appropriate caution and consult the product insert for further information If a product insert does not exist or if the product insert does not contain the symbol or the required information consult the user manual EN Indicates the presence of an electrical shock hazard and to proceed with appropriate caution AN Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution 238 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix G Safety G Instrumentation safety Description Indicates the presence of a pinching hazard and to proceed with appropriate caution
135. alization Plate with VIC ROX Dye Kit TaqMan RNase P 96 Well Instrument Verification Plate 4432382 s TaqMan RNase P 96 Well Instrument Verification Plate Fast 96 well sample block kits QuantStudio 12K Flex System consumable Part no STE Fast 96 Well Spectral Calibration Plate with FAM Dye 4432389 15 to 25 Fast 96 Well Spectral Calibration Plate with VIC Dye 4432396 Fast 96 Well Spectral Calibration Plate with ROX Dye 4432402 Fast 96 Well Spectral Calibration Plate with SYBR Green Dye 4432408 Fast 96 Well Spectral Calibration Plate with TAMRA Dye 4432414 Fast 96 Well Spectral Calibration Plate with NED Dye 4432420 Fast 96 Well Region of Interest ROI and Background Plates 4432426 e Fast 96 Well Region of Interest ROI Calibration Plate e Fast 96 Well Background Plate Fast 96 Well Normalization Plates with FAM ROX and VIC 4432432 ROX Dyes e Fast 96 Well Normalization Plate with FAM ROX Dye e Fast 96 Well Normalization Plate with VIC ROX Dye Kit TaqMan RNase P Fast 96 Well Instrument Verification Plate 4351979 s TaqMan RNase P Fast 96 Well Instrument Verification Plate 234 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix F Parts and Materials E Calibration and verification kits Array card sample block kits QuantStudio 12K Flex System consumable Part no Storage C Array Card Spectral Calibratio
136. alse Baseline Start The first cycle used to calculate the baseline Float Baseline End The last cycle used to calculate the baseline Float Custom1 Custom6 The contents of the custom text fields found in the Results table of the experiment 1024 character string per field Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix E File Format Reference E 7900 export format 7900 export format Exportable files Setup file File header The QuantStudio 12K Flex Software can export setup and results data from experiment files eds to tab delimited text files txt in a legacy export format of the Applied Biosystems 7900HT Real Time PCR System The 7900 export format features a standardized data structure and markup to maximize accessibility by downstream applications Data exported in the QuantStudio12KFlex export format can be opened by common spreadsheet applications such as Microsoft Excel or imported by laboratory information management system LIMS applications that have been configured to parse the file format Note Due to the very different nature of the QuantStudio 12K Flex Instrument some export types are not available Note Column customization sorting and omission is not available Only multiple tab delimited
137. ample Block 4453553 QuantStudio 12K Flex System 96 Well Sample Block 4453556 QuantStudio 12K Flex System Array Card Sample Block 4453554 QuantStudio 12K Flex System Fast 96 Well Sample Block 4453559 QuantStudio 12K Flex System OpenArray Sample Block 4471025 232 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix F Parts and Materials E Calibration and verification kits Calibration and verification kits 384 well sample block kits The following kits are to be used with the Applied Biosystems QuantStudio 12K Flex Real Time PCR System The following materials are required to calibrate the QuantStudio 12K Flex System S 384 well sample block kits 0 eens see below R 96 well sample block Kita 234 m Fast 96 well sample block kits nuunuu ranura nets 234 m Array card sample block kits n eee ees 235 Note For reagent or consumable shelf life expiration date see the package label QuantStudio 12K Flex System consumable Part no Seo 384 Well Spectral Calibration Plate with FAM Dye 4432271 15 to 25 384 Well Spectral Calibration Plate with VIC Dye 4432278 384 Well Spectral Calibration Plate with ROX Dye 4432284 384 Well Spectral Calibration Plate with NED Dye 4432302 384 Well Spectral Calibration Plate with SYBR Green Dye 4432290 384 Well Spectral Calibration Plate with TA
138. an The average C of the replicate wells for the Float specified target sample combination Ct SD The standard deviation of the average Cy of the Float replicate wells for the specified target Number of The number of quality flags generated by the Integer Flags sample during the analysis Tm Melting point Float Tm1 Tm3 The first second and third melting temperatures Float Tm calculated in degrees Celsius Comments Additional text that describes the well 1024 character string Custom The contents of the custom text fields found inthe 1024 characterstring Custom Results table of the experiment per field If analysis flags are present results data is present in additional columns named for the associated flags true Or false Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 221 File Format Reference QuantStudio12KFlex export format Presence absence The following table describes the results data exported from presence absence experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 chara
139. and Administration User Guide Ordering from the 1 QuantStudio 12K Flex Software Appendix F Parts and Materials How to order To find your assay on the Life Technologies Store complete the Find Assay pane in the QuantStudio 12K Flex Software a Enter a gene name in the Enter Gene Name field then click Find Assay b In the Find Assay Results dialog box select your assay c Click Apply Assay Selection The selected assay gets added to your shopping list Check that the Experiment Shopping List contains the desired materials other than the assay selected in the previous step and that the quantities are correct then click Order Materials in List In the Order Materials Login dialog box enter your user name and password for the Life Technologies Store then click Log In and Submit x Log into the Applied Biosystems Store to place the selected items in your shopping basket If you do not have a user name and password click Register Now to create a new account Store Log In OR Register To log into the Applied Biosystems Store enter your user name you do not and password then click Log In and Submit have an Applied Biosystems User Name FT account click the link below to eate Password l l create anew account Log In and Submit Register Now T Remember my user name and password for future orders Note If you do not have an account with the Life Technologies Store click
140. and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials e Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition http www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en 248 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Safety alerts General alerts for all chemicals General alerts for instrumentation Specific alerts for instrumentation Appendix G Safety G Safety alerts Avoid contact with skin eyes and or clothing Read the SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CAUTION Before using a cleaning or decontamination method other than those recommended by the Life Technologies verify with Life Technologies that the proposed method will not damage the equipment T WARNING This instrument is designed for 12 V 75 W halogen LED only CAUTION FIRE HAZARD For continued protection against the risk of fire replace fuses only with listed and cert
141. and Materials 0 0 e eee eens 229 How toorder TTT 2 patente ple Ee hls Beg Misia Medias tee eo 230 Ordering from the QuantStudio 12K Flex Software 2 2 0 00 0 cece cece eee eee 231 Ordering from the Life Technologies Website cece eee 231 ACCOSSONIES 2 a gt ben neskie adhe ss wade nite ee ue dose RS nie ee eee 232 Calibration and verification Kite 0 0 0 c cece eect ttt eee eee 233 384 well sample block Kite 233 96 well sample block dte 234 Fast 96 well sample block KLS 234 Array card sample block kits nanunua ennn nnne rrenaren 235 Consumables T A S Rate ois RAS ote te Fits ee PES PS ste Sea AT 236 10 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Contents APPENDIX G Safety ames bawadeaen tees 237 Instrumentation safety srediti aa A Z cece E EE A aE eee e TART 238 Symbols oninstruments 0 0000 cece ee eee cee aranera 238 Locations of safety labels on instruments 00000 0c eee e eee eee 240 General instrument eae 0 000 cece cece ee eeeees 241 Physicalihazard safety ints ase ce Aether ec dette a a ad head E cee 242 ElecthicalSatety nsare anata ate D seis Ak ee ee E A 242 Bar code scanner laser Safety 000 cece eee ence eens 243 Workstation Safety is sen ees kl een dea eh tee ee ee ceo ee RE ees 243 Safety and electromagnetic compatibility EMC standards 0 0 eee ee 244 Chemical Satetys dei
142. and computer power receptacle on an electrical circuit that is not shared with electrically noisy devices or devices that can cause power surges such as refrigeration units The following table provides electrical specifications for the instrument and associated devices For all indicated input voltages a 15 A circuit is required Rated Rated Rated Rated Device current power voltage frequency QuantStudio 12K Flex 12 5A 950 VA 100 240 50 60 Hz Instrument 10 VAC Computer 2 1A 125 VA Monitor 1 5A 65 VA Twister Robott 2 5A 150 VA OpenArray Accufill System 0 6 A 75VA Optional component of the QuantStudio 12K Flex System Note The instrument monitor desktop computer Twister Robot and laptop computer self adjust for 100 240V input voltages of 50 60 Hz Environmental requirements Requirement Description Altitude Less than 2000 m 6500 ft above sea level Temperature 15 30 C 59 86 F Do not place the QuantStudio 12K Flex Instrument next to heaters cooling ducts or in direct sunlight Temperature fluctuations can affect performance Humidity QuantStudio 12K Flex Instrument computer and the UPS unit 20 80 noncondensing OpenArray Accufill System maximum humidity e 80 at 31 C e 50 at 40 C Pollution The instrument has a pollution degree rating of II The noise output of the instrument is lt 60 dB at idle
143. and read between 505 723 nm Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 19 1 Getting Started Specifications and layout Specifications and layout QuantStudio 12K The figures below summarize the specifications and requirements for the Flex System QuantStudio 12K Flex System For more information refer to the Applied Biosystems specifications QuantStudio 12K Flex Real Time PCR System Site Preparation Guide Part no 4470654 Component Height Depth Width Weight QuantStudio 12K Flex 73 8 cm 66 0 cm 50 4 cm 70 0 kg Instrument 29 0 in 26 0 in 19 8 in 154 3 lbs Computer 56 5 cm 54 7 cm 21 6 cm 24 9 kg 22 3 in 22 4 in 8 5 in 55 0 lbs Monitor 38 0 cm 13 7 cm 37 4 cm 3 0 kg 15 0 in 5 4 in 14 7 in 6 7 lbs Keyboard 5 0 cm 15 25 cm 44 7 cm 0 1 kg 2 0 in 6 0 in 17 5 in 0 2 lbs OpenArray Accufill System 76 0 cm 64 0 cm 79 0 cm 55 0 kg 30 0 in 25 0 in 31 0 in 120 0 lbs Twister Robot 97 0 cm 71 0 cm 52 0 cm 52 2 kg 38 0 in 28 0 in 20 5 in 115 0 lbs Weight varies depending on the sample block installed Computer specification differs depending on the computer ordered with the QuantStudio 12K Flex System laptop or desktop Optional component of the QuantStudio 12K Flex System Figure 1 QuantStudio 12K Flex System with Twister Robot 15 2 cm 6
144. antStudio 12K Flex System 6 66 18 mM Specifications and Java s iieu ER Tag T ER a EET a NTE RE RT T 20 S QuantStudio 12K Flex System hardware 0 00 ccc e eee e eee 24 S QuantStudio 12K Flex System Software nananana rreren rerne 30 S QuantStudio 12K Flex System consumables 0 00 e cece eee eee 32 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 17 1 Getting Started About the QuantStudio 12K Flex System About the QuantStudio 12K Flex System The Applied Biosystems QuantStudio 12K Flex Real Time PCR System uses fluorescent based polymerase chain reaction PCR reagents to provide e Quantitative research detection of target nucleic acid sequences targets using real time analysis e Qualitative research detection of targets using post PCR endpoint analysis e Qualitative analysis of the PCR product achieved by melt curve analysis that occurs post PCR About data The Applied Biosystems QuantStudio 12K Flex Real Time PCR System collects raw collection fluorescence data at different points during a PCR depending on the type of run that the QuantStudio 12K Flex Instrument performs Run type Data collection point Real time Standard curvet The QuantStudio 12K Flex Instrument collects data following each extension step of the PCR Relative standard curvet Comparative Cy AAC7 Mel
145. antStudio 12K Flex Used to calibrate and perform experiments on the Software QuantStudio 12K Flex Instrument QuantStudio 12K Flex Used to automate the creation of new experiments System Command line and the export of existing experiments Utility SampleTracker Software Used to rapidly enter sample information into OpenArray experiments ExpressionSuite Software Analyzes gene expression data generated by the QuantStudio 12K Flex Instrument AccuFill Software Controls the AccuFill instrument used to load OpenArray plates HRM Software Module An optional QuantStudio 12K Flex Software module that allows you to set up run and analyze an high resolution melting curve experiment TaqMan Genotyper Analyzes genotyping data generated by the Software QuantStudio 12K Flex Instrument Twister Robot Software Controls the Twister Robot stores all of the taught positions for the robot and includes the Visual Basic code required to operate the Twister Robot with the automation control software T We recommend that you do not install programs to the C drive Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Twister Robot software Third party software Chapter 1 Getting Started 1 QuantStudio 12K Flex System Software The Twister Robot Software consists of several applications that are used to calibrate program and op
146. ard 1024 character string Comments The additional text that describes the well 1024 character string After the assay data the QuantStudio 12K Flex Software exports the well data that describes the configuration of samples and assays on the experiment consumable The table below describes the well data that can be exported from absolute quantification relative quantification or presence absence experiments If a well contains more than one assay the QuantStudio 12K Flex Software lists the setup data for each additional assay in additional columns to the right of the existing data Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 character string Detector The name of one target assay applied to the well 100 character string Name if applicable Task Task the target is used for in this well UNKNOWN STANDARD or NTC Quantity The standard quantity if applicable This Float or Integer column only appears for Standard Curve and Relative Standard Curve experiments Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom 224 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix E File Format Reference E 7900 export format Multicomponent The QuantStudio 12K Flex Software can export
147. ard kit Repeat the experiment and make sure to properly seal and centrifuge the RNase P plate or array card If the replacement RNase P plate or array card fails contact Life Technologies for further assistance Well displays the THOLDFAIL flag indicating that the software cannot calculate the threshold e Amplification too early e Amplification too late e Low amplification e No amplification If a well is flagged confirm the results 1 2 Select the flagged well s in the plate layout or well table View the amplification plot Rn vs Cycle and ARn vs Cycle and check for early late low or no amplification Contact Life Technologies to order a replacement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Life Technologies for further assistance Instrument malfunction Multiple possible causes Contact a local Life Technologies Field Service Office sy20 g ajdues pueg Aewy pue aed N M MNW Bunewqneg Z 4aydeyg 6unooysayqnos E Calibrating Multi Well Plate and Array Card Sample Blocks Troubleshooting Identifying contamination Signals that exceed the limit of normal background fluorescence may indicate fluorescent contaminants on the calibration plate or the sample block Common contaminants include ink residue from permanent pens powder from disposable gloves and dust To determine the source and location of the contamination
148. array cards for instrument calibration sasan c cece eee ee eee eee eee 37 Required materials dai etain ani df aid aes och lead getiag 37 Filling the calibration array cards cece ees 37 ROI calibration i 69T S A cle cated deans Cable Polen Gable Puede ea eek eed Sah ed ee 41 When to perform the calibration 0000 0 ccc eee eee eee eee 41 About the ROI calibration data 2 K a e DA eZ ERa H E KRE EE 03 41 Preparing the calibration plate or array card 000 00ers 42 Preparing the ROI calibration plate nannan anaren 42 Performing the calibration 0 0c rennene 43 Background calibration 2 0c c cece eeraa 45 When to perform the calibration 0000 cece eee eee eee eee eee 45 About the background calibration data 0 ccc eee eee eee ee 45 Preparing the calibration plate or array Card cee eee eens 45 Preparing the background plate 000 c eee eee eee eae 46 Performing the calibration cece eee eee neces 47 Uniformity calibration eesi ei weeds H a Ea bgt ZARE eel ieee 49 When to perform the calibration 000 c eee eee eee eee eee 49 About the uniformity calibration data saasaa sauran arrra enrere 49 Preparing the calibration plate or array card 0000 e eee eee eee eens 49 Preparing the calibration plate annann anuanua annerer aeaea 50 Performing the calibration 000 cece eee ee eee eee ees 51 Dyeicalibrationt tt
149. at the Float well Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom RDML export format For more information 228 The QuantStudio 12K Flex Software can export data from real time quantitative PCR experiments as well formed Real time PCR Data Markup Language RDML a structured extensible markup language XML standard for quantitative PCR qPCR data In combination with the Minimal Information MIQPCR guidelines the RDML element structure describes all aspects of a qPCR experiment including setup analysis and data interpretation The exported RDML data is saved as a flat text file that can be used to transfer qPCR data between the QuantStudio 12K Flex Software and third party applications IMPORTANT The RDML export format is available only for standard curve gene expression and relative standard curve experiments The RDML standard is maintained by the RDML consortium an organization that consists of key developer groups and a member community For more information on the RDML format visit the RDM organization website www rdml org The website features free data management tools including an on line RDML file generator and RDML software libraries Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Parts and Materials This appendix covers E How to ordetisc2 sian vhasa eae ead
150. ated cover be sure to wait until it reaches room temperature Required materials e Safety glasses e Powder free gloves e Plate adapter Replacing the plate IMPORTANT If you are installing a plate adapter of a different format you may also be adapter required to change the sample block to match the new consumable format 1 Touch the instrument touchscreen to activate it then press Q 2 In the Main Menu touch k 3 When the tray arm opens pull the latch then lift and remove the plate adapter 4 Attach the new adapter to the tray arm then pull the latch to allow the adapter to lower into place If necessary apply pressure as indicated until the adapter snaps into place 5 In the Main Menu touch g 6 Ifyou are installing a tray adapter of a different consumable format replace the sample block if necessary Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 123 4 Maintenance Replacing the plate adapter 124 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Networking This chapter covers M Networking Overview 6 6 6 c ce eens 126 S Network setup workflow e eee eens 128 m Collecting the required network information 00 cece eee ee eee 129 m Connecting the QuantStudio 12K Flex Instrument to the network 129 m Connecting the computer to the network ees 130 S Monitor
151. ation User Guide 45 Calibrating Multi Well Plate and Array Card Sample Blocks Background calibration Prepa ring the IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or background plate array cards Required materials e 96 or 384 Well Region of Interest ROD and Background Plates e Centrifuge with plate adapter e Powder free gloves e Safety goggles Note Only the background plate is required for this calibration Preparing the calibration plate 1 Remove the background plate from the freezer then allow it to warm to room temperature approximately 5 minutes 2 Remove the background plate from its packaging Do not remove the optical film IMPORTANT Do not discard the packaging You can use the background plate to calibrate a QuantStudio 12K Flex System 3 times for up to 6 months if it is stored in its original packaging sleeve 3 Vortex and centrifuge the background plate a Vortex the background plate for 5 seconds b Centrifuge the plate for 2 minutes at lt 1500 rpm IMPORTANT The background plate must be well mixed and centrifuged c Confirm that the liquid in each well of the background plate is at the bottom of the well If not centrifuge the plate again at a higher rpm and for a longer period of time IMPORTANT Do not allow the bottom of the plate to become dirty Fluids and other contaminants that adhere to the plate bottom can contaminate the sampl
152. audit users enter reason for change 155 overview 154 AutoDelta 254 Autodiscovery instrument 170 automatic baseline 254 automatic threshold 254 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 269 Index background calibration 45 84 254 data 45 84 perform 47 troubleshoot 73 when to perform 45 84 background fluorescence 45 backup experiment files 110 instrument settings 110 164 barcode file about 191 format 207 barcode readers 26 27 barcodes supported 26 baseline 254 baseline corrected normalized reporter DRn 255 biohazardous waste handling 248 biological replicate 255 blocked IPC 255 C calibration array cards 32 37 background 45 84 consumables 32 236 custom dye 184 dye 53 90 kits 233 normalization 59 plaque 83 plates 32 reminders enable disable 134 ROI 41 uniformity 49 87 calibrator 255 CAUTION description 14 chemical safety 245 chemical waste safety 246 247 clearances instrument components 21 required 21 command line application command syntax and arguments 194 196 running 193 comparative CT method 255 compatibility third party software 31 computer experiment files maintenance 110 hard drives maintenance 110 remote monitoring 130 132 requirements 30 connections 23 consumables 236 contamination identification 80 107 sample block decontamination 111 control instrument over a network 126 create array cards for calibratio
153. auditing globally and by record type It is disabled globally by default e Electronic signature e sig Determines if users are required to provide a user name and password when performing certain functions You can configure e sig so that a user can print a report or start a run only if the associated data are signed You can also configure each e sig event to require multiple signatures and to require users with specific permissions to sign Note Electronic signature can be enabled or disabled globally It is disabled globally by default You can configure the SAE module in a variety of ways For example you can e Require users to log in and leave audit disabled e Allow only certain users to create or modify protocols e Allow only certain users to approve reviewed samples e Require experiments to be signed before users can run or print them Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 6 Security Audit and Electronic Signature 6 Configuring the system security Configuring the system security Accessing the Use the Security screen to disable and enable security control restrictions and security Security screen policies for all user accounts and set up notifications when certain security events occur and enabling or disabling security IMPORTANT If you disable security you inactivate audit and electronic signature functions however no audit re
154. ause some wells to be amplified insufficiently or not at all These wells typically produce Cy values that differ significantly from the average for the associated replicate wells If included in the calculations these outlying data outliers can result in erroneous measurements 8 In the Amplification Plot select Ct vs Well from the Plot Type menu then verify the uniformity of each replicate population controls standards and unknowns on the reaction plate by comparing the groupings of Cy values a In the plate layout select the wells containing Unknown Population A e 96 well plate Select rows A C 5 000 copy population e 384 well plate Select columns 7 15 5 000 copy population e Array card Select rows C and D 800 copy population b In the plot verify that the Cy values of the replicate population are equivalent Note The numbers on the X axis of the plot correspond to the wells of the reaction plate Beginning with well A1 the wells are numbered from left to right and top to bottom c If an outlier is present in the selected population select the corresponding well of the plate layout then click Omit to remove the well from the analysis If the total number of outliers for the replicate population exceeds the limit in the table below repeat the experiment using another RNase P plate or array card Maximum number of outlier wells that can be removed Sample block Standard N l t
155. aw at room temperature approximately 15 minutes Note Unopened OpenArray plates can remain at room temperature for up to 24 hours 3 Prepare a syringe containing OpenArray Immersion Fluid Attach the syringe tip to the syringe then set the assembly on a clean surface IMPORTANT The application of the syringe tip requires force Confirm that the tip is locked firmly in place before proceeding OpenArray Immersion Fluid Cap remove a Syringe tip attach Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 3 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance 4 Pipet 5 0 uL of the RNase P solution into loading position 1 of the 384 well sample plate 10 12 14 16 18 20 22 OpenArray 1 SB882Z 820 HE GE GE RNase D ASN Iw l ael G IE FR EP a uP K TAT Tw L l F 5 Cover the sample plate with a foil seal then score or cut the foil into the 8 sections shown above 6 Centrifuge the plate for 1 minute at 1500 rpm then place the plate on ice to keep the samples cold Initializing the 1 Close the enclosure door then start the OpenArray Accufill software The system software checks the computer and connections as the system starts When prompted clear the deck and empty the waste bin of used tips a Open the instrument by grasping the enclosure door handle and gently but firmly
156. ays the aligned data as the difference in fluorescence between the reference curve and the other melt curves 262 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide reference sample refSNP ID region of interest ROI calibration regression coefficients regression line reject well relative standard curve method Relative Threshold algorithm relative threshold cycle Cpr replicate group replicates Glossary In relative standard curve and Comparative Cy AAC experiments the sample used as the basis for relative quantification results Also called the calibrator The reference SNP refSNP cluster ID Generated by the Single Nucleotide Polymorphism Database of Nucleotide Sequence Variation dbSNP at the National Center for Biotechnology Information NCBI The refSNP ID can be used to search the Life Technologies Store for an Applied Biosystems SNP Genotyping Assay Also called an rs number Type of calibration in which the software maps the positions of the wells on the sample block of the instrument The software uses the ROI calibration data to associate increases in fluorescence during a run with specific wells of the plate A calibration image for each individual filter must be generated to account for minor differences in the optical path Values calculated from the regression line in standard curves including the R2 value slope and y intercept Y
157. cannot cancel a report after you click a view button Click View Report In the Report screen click tool bar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item e To print the report click Print e To save the report electronically PDF click H Save Close the report Archiving purging and restoring audit records The audit archive function makes a copy of audit records Purge makes a copy of audit records and then deletes them You can use the Restore function to restore purged audit records Action Procedure Archive To selectively archive or purge delete system configuration or action audit and purge records 1 Select the System Configuration History tab 2 Select records in the appropriate screen 3 Click Archive or Purge 4 If you select Archive specify a location and name for the archive file asz Restore Te O restore system configuration or action audit records click Restore then select the ASZ file to restore 148 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 6 Security Audit and Electronic Signature Generating audit reports Exporting audit records You can export audit records to a txt file for additional manipulation and reporting outside the QuantStudio 12K Flex Software 1 Display the records of interest as described above 2
158. cates whether smoothing is turned on for the experiment true Or false Stage Cycle where Analysis is performed The stage and cycle during the thermal cycling protocol when the QuantStudio 12K Flex Instrument collected data Stage lt integer gt Step lt integer gt Calibration Date Calibration Expiration Date The date and time that the current background ROI uniformity or pure dye calibration was performed and when it will expire lt date and time gt lt date and time gt Instrument serial number The serial number of the QuantStudio 12K Flex Instrument that ran the experiment lt 100 character string gt Quantification cycle method The method of quantification for the associated experiment lt 100 character string gt 210 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Sample setup data Appendix E File Format Reference E QuantStudio12KFlex export format When selected as an export option the QuantStudio 12K Flex Software exports sample setup data after the file header The sample setup data describes the sample configuration on the experiment consumable including positions sample names task assignments assay information and color coding The data consists of a column header followed by the sample data fields where each row contains the data for a single well separated by tab characters If
159. ce then click Dye b In the Dye Calibration screen select System Dye Calibration then click Start Calibration 5 In the Dye Calibration screen select the dye to calibrate from the Dye Name drop down list then perform the calibration as instructed 6 Load the calibration plate or array card into the QuantStudio 12K Flex Instrument a Confirm that the dye plate or array card that you are about to load matches the dye selected in the QuantStudio 12K Flex Software The name of the dye contained by the consumable is next to the barcode on the front of the plate or array card b Load the dye plate or array card into the plate adapter Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter s B Load both plates and array cards with the barcode facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 7 After loading the plate or array card start the calibration a In the Dye Calibration screen select Check the box when the dye calibration plate has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Not
160. ce experiments the level of fluorescence above which the software assigns a presence call The PCR cycle number at which the fluorescence meets the threshold in the amplification plot See melting temperature Tm Instrument display that you touch to control the instrument Type of calibration in which the software measures sample block uniformity The calibration generates data that compensate for the physical effects of the QuantStudio 12K Flex System filters on data collected during an experiment In the software the task for the target or SNP assay in wells that contain the sample being tested In quantification experiments the task for the target in wells that contain a sample with unknown target quantities In genotyping experiments the task for the SNP assay in wells that contain a sample with an unknown genotype In presence absence experiments the task for the target in wells that contain a sample in which the presence of the target is not known In melt curve experiments the task for the target in wells that contain a sample with an unknown melt curve profile In presence absence experiments wells that contain a sample and internal positive control IPC In the standard curve the value of y where the regression line crosses the y axis The y intercept indicates the expected threshold cycle Cy for a sample with quantity equal to 1 268 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and A
161. ch the QuantStudio 12K Flex System network settings touchscreen to activate it then press G 2 In the Main Menu touch Settings then touch Set Network Information Note The Set Network Information screen displays the Media Access Control MAC address of the QuantStudio 12K Flex Instrument below the Autodiscovery and Smart Monitoring check boxes The MAC address can be used to uniquely identify the QuantStudio 12K Flex Instrument on the network 3 Touch Autodiscovery to make the QuantStudio 12K Flex Instrument discoverable by computers that are running the QuantStudio 12K Flex Software 4 Touch Smart Monitoring to enable the feature on the QuantStudio 12K Flex Instrument The Smart Monitoring feature allows Life Technologies service personnel to monitor the status of the QuantStudio 12K Flex Instrument remotely through an internet connection Smart Monitoring employs multiple layers of security including a Secure Sockets Layer SSL and Lightweight Directory Access Protocol LDAP authentication to provide real time troubleshooting and problem resolution for the QuantStudio 12K Flex Instrument For a detailed description of the Smart Monitoring Service see the Smart Monitoring Service Product Bulletin Leveraging the power of the Internet while maintaining system security Part no 121PB07 03 5 Set the Internet Protocol TCP IP Properties for either DHCP or Static IP communication PS k Action s
162. cord is generated to indicate that audit and electronic signature functions are disabled Note Security is enabled by default To enable or disable security 1 In the QuantStudio 12K Flex Software select Tools gt Security gt Settings 2 In the Security Settings dialog box select the System tab 3 Select or deselect Enable Security Note the following e Disabling Security inactivates Auditing and E Signature e The enable commands are grayed when a run is in process e When security is disabled the is not active in lower parts of the screen e The software requires you to enter your user name and password when you enable security IMPORTANT If you enable or disable the QuantStudio 12K Flex Software security auditing and electronic signature feature you must similarly enable or disable the QuantStudio 12K Flex Instrument security see page 172 The QuantStudio 12K Flex Software cannot connect to QuantStudio 12K Flex Instruments that do not match security settings 4 Click Apply Settings Setting the account Note Security policies apply to all user accounts and security 1 lici In the QuantStudio 12K Flex Software select Tools gt Security gt Settings policies 2 In Account Setup specify the user name limits TM IMPORTANT The QuantStudio 12K Flex Software allows spaces in user names Use spaces in user names with caution For information see Spaces in user names and or passwo
163. cter string Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN Or NTC Reporter The reporter dye that labels the probe for the 100 character string target assay Quencher The quencher dye that labels the probe for the 100 character string target assay Rn The raw fluorescence for the associated well Float normalized to the fluorescence of the passive reference dye Rn Mean The averaged normalized fluorescence R for the Float associated replicate wells that contain the same target sample combination Rn SD The standard deviation of the normalized Float fluorescence R for the associated replicate wells that contain the same target sample combination Threshold The calculated value of the threshold for a positive Float Value call Call The presence absence call assigned tothe sample Negative Control at the specified well Blocked IPC Control TPC Failed Positive or Negative Comments Additional text that describes the well 1024 character string Automatic Ct Threshold Indicates whether the threshold was determined automatically true or manually false true Or false Ct Threshold The threshold cycle C7 for the sample at the well Float Automatic Ct Indicates whether the baseline was determined true Or false Baseline automatically true or manually f
164. dditional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 245 C Safety Chemical safety SDSs Chemical waste safety About SDSs Chemical manufacturers supply current Safety Data Sheets SDSs with shipments of hazardous chemicals to new customers They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated SDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new SDS packaged with a hazardous chemical be sure to replace the appropriate SDS in your files Obtaining SDSs The SDS for any chemical supplied by Life Technologies is available to you free 24 hours a day To obtain SDSs 1 Go to www lifetechnologies com click Support then select SDS 2 In the Keyword Search field enter the chemical name product name SDS part number or other in
165. ding to the Real time PCR Data Markup Language RDML data standard CT and CRT are the algorithm specific calculations of C Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 261 Glossary quantification method quantity quencher QuickStart R2 value ramp ramp speed raw data plot reaction mix reagents real time PCR Real time PCR Data Markup Language RDML reference In quantification experiments the method used to determine the quantity of target in the samples In quantification experiments the amount of target in the samples Absolute quantity can refer to copy number mass molarity or viral load Relative quantity refers to the fold difference between normalized quantity of target in the sample and normalized quantity of target in the reference sample A molecule attached to the 3 end of TaqMan probes to prevent the reporter from emitting fluorescence signal while the probe is intact With TaqMan reagents a nonfluorescent quencher minor groove binder NFQ MGB can be used as the quencher With SYBR Green reagents no probe and therefore no quencher is used A feature that allows you to run an experiment without entering plate setup information if your instrument and computer are in the same network QuickStart requires an experiment template file Regression coefficient calculated from the regression line in the standard curve
166. dministration User Guide Numerics 128 ASCII character barcode support 26 7900 export file format 223 A accessories 232 account setup 139 suspended activate 142 suspension 139 154 user 142 action log contents 148 display 146 149 152 activation license keys 117 administrator auditing 145 password 138 security 138 user role 143 AIF 253 AIF 253 AIX 253 allele 253 allelic discrimination plot 253 altitude requirement 22 amplicon 253 amplification 253 amplification efficiency EFF 253 amplification plot 253 amplification stage 253 Analysis Protocol Library 253 annual maintenance tasks 36 82 APIPA support 126 archive audit records 148 experiment files 110 instrument settings 110 164 Index arguments command line batch file creation 194 results exportation 196 array card background creating 182 calibration 32 prepare for calibration 37 prepare for verification 37 66 Array Card RNase P Kit 64 233 assay 254 Assay ID 254 assay index file 253 assay information file 191 254 file format 207 audit administrators 145 action log 146 148 149 152 archive records 148 audit actions 148 audit mode 145 audit reason settings 145 audited objects and actions 145 enable or disable 145 export records 149 export settings 153 import settings 153 object audit history 146 149 152 overview 138 purge records 148 restore records 148 system configuration history 146 147 149 152 when security is disabled 145
167. e The header contains the lines listed in the following table Field Description Output Block Type The model of the sample block installed to the QuantStudio 12K Flex Instrument at the time the experiment was run 96 384 well Of array card Calibration Expired Expiration status of the calibration Indicates whether the calibration of the QuantStudio 12K Flex Instrument was current at the time that the experiment was run Yes Or No Chemistry The chemistry of the experiment lt 100 character string gt Experiment File Name The path to the experiment file on the local computer hard drive lt filepath gt Experiment Name The name of experiment entered into the Experiment Name field lt 100 character string gt Experiment Run End Time The date and time that the QuantStudio 12K Flex Instrument finished running the experiment lt date and time gt Experiment Type The type of chemistry application for which the experiment is designed Standard Curve Presence Absence Relative Standard Curve Or DDCt Quantification Instrument Type The model of the QuantStudio 12K Flex Instrument that ran the experiment QuantStudiol2KFlex Passive Reference The dye used as a passive reference or blank if the consumable did not contain one lt 100 character string gt Signal Smoothing On The smoothing setting status for the experiment Indi
168. e U e X U e Failing image Red circles appear around some or none of the wells indicating that the wells did not calibrate The absence of a circle indicates that the region of interest for the well position is not sufficiently bright Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 41 Calibrating Multi Well Plate and Array Card Sample Blocks ROI calibration Preparing the calibration plate or array card Preparing the ROI calibration plate IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or array cards Prepare the ROI calibration consumable appropriate for your QuantStudio 12K Flex Instrument E Preparing the ROI calibration plate 6 eee 42 E Preparing array cards for instrument calibration 0 00 e eee eee 37 Required materials 96 or 384 Well Region of Interest ROI and Background Plates Centrifuge with plate adapter Powder free gloves Safety goggles Note Only the ROI plate is required for this calibration Preparing the calibration plate 1 3 Remove the ROI calibration plate from the freezer then allow it to warm to room temperature approximately 5 minutes IMPORTANT Do not remove the calibration plate from its packaging until you are ready to run it The fluorescent dyes in the wells of the plate are photosensitive Prolonged exposure to light can dimi
169. e Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature 56 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Dye calibration 8 When the run is complete and the QuantStudio 12K Flex Software displays the Analysis screen confirm the grouping of the dye spectra a Select the Plate Layout tab then review the raw data For each spectrum verify that the peak is e Within the detectable range for the QuantStudio 12K Flex System e Free of irregular spectral peaks e Present at the correct filter for the dye see the following table Filter set Excitation nm Emission nm System dyes x1 m1 Blue 470 15 520 15 s FAM dye s SYBR Green dye x2 m2 Green 520 10 558 12 s HEX dye e JOE dye s TET dye s VIC dye x3 m3 Yellow 549 5 10 586 5 10 s NED dye e TAMRA dye x4 m4 Orange 580 10 623 14 ROX dye x5 m5 Red 640 10 682 14 LIZ dye x6 m 6 Deep red 662 10 711412 t T No Life Technologies fluorescent dyes are collected at the x6 m filter set Note Among wells containing the same dye variations in spectral position and peak position are caused by minor differences in the optical properties and excitation energy between the individual wells b
170. e lt 1024 character string gt Custom sample in the well Well Sample Name Customl Custom2 Custom3 Custom4 Custom5 Custom6 21 Sample ltestl test2 test3 test4 test5 test 22 Sample 2testl test2 test3 test4 test5 test 23 Sample 3testl test3 test4 test5 test 1 Sample 5testl test2 test3 test4 test test 2 Sample 6testl test2 test3 test4 test test 3 Sample Ttestl test2 test3 test4 test test 4 Sample 8testl test2 test3 test4 test test 206 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Barcode file format Appendix E File Format Reference Barcode file format The QuantStudio 12K Flex Software command line application can import barcode files to populate experiment files eds it generates with barcode information A barcode file is a tab delimited ASCII text file txt that contains a list of barcodes The files can be created manually using a text processor or generated automatically by third party applications IMPORTANT To guarantee successful import the file must contain all the elements described in the following section and in the order that they appear File structure The barcode file contains a list of barcodes where each line defines a single barcode terminated by a carriage return The barcodes can occur in any order and cannot contain starting or trailing white space Note The QuantStudio 12K Flex Software command line application doe
171. e the heat seal on the plate may be defective resulting in the evaporation of the associated samples 4 Contact Life Technologies to order a replacement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Life Technologies for further assistance Instrument does not eject the Adhesive cover may have 1 Power off the QuantStudio 12K Flex Instrument RNase P plate adhered the plate to the heated 2 wait for 15 minutes then power on the QuantStudio 12K Flex Instrument and eject cover within the instrument the pl plate 3 If the plate does not eject power off and unplug the QuantStudio 12K Flex Instrument then open the access door 4 Wearing powder free gloves reach into the QuantStudio 12K Flex Instrument and remove the plate from the heated cover then close the access door 5 Perform a background calibration to confirm that the sample block has not been contaminated Well displays the NOSIGNAL flag indicating that the well produced very low or no fluorescence signal Missing reaction mix resulting from pipetting error If a well is flagged confirm the results 1 2 Consider omitting the well from the analysis Note the location for each flagged well and check each corresponding well in the reaction plate for evaporation or low reaction volume Contact Life Technologies to order a replacement TaqMan RNase P plate or array card kit If t
172. e OpenArray case by the edges s Do not touch the through holes of the OpenArray plate e Load and seal a OpenArray plate within one hour after opening the packaging e Ifyou drop a loaded OpenArray plate discard it in the appropriate waste container Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 95 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance Required materials s QuantStudio 12K Flex System RNase P Kit including OpenArray RNase P Reaction Mix QuantStudio 12K Flex System OpenArray Lid QuantStudio 12K Flex System OpenArray Plug QuantStudio 12K Flex System OpenArray Immersion Fluid QuantStudio 12K Flex System OpenArray Immersion Fluid Tip OpenArray Digital PCR Plate OpenArray 384 Well Sample Plate e OpenArray AccuFill System e OpenArray Plate Press e Bleach 10 e Ethanol e OpenArray 384 Well Sample Plates e OpenArray AccuFill System Loader Tips e Pipettes e Powder free gloves e Safety glasses Preparing for the IMPORTANT Wear powder free gloves while preparing the OpenArray plates verification expe riment 1 Confirm that the OpenArray 384 well sample plate OpenArray AccuFill 96 Loader Tips and plate holder are completely clean and dry 2 Remove an OpenArray plate from the freezer but do not open the packaging Allow the plate to th
173. e QuantStudio 12K Flex Software uses the ROI calibration data to associate increases in fluorescence during a run with specific wells on the plate The QuantStudio 12K Flex Instrument uses a set of optical filters to distinguish the fluorescence emissions gathered during runs You must generate a calibration image for each filter to account for minor differences in the optical path Perform the ROI calibration every year or as often as necessary depending on instrument use IMPORTANT After every ROI calibration you must perform a background calibration uniformity calibration dye calibration normalization calibration and RNase P instrument verification experiment During the ROI calibration the QuantStudio 12K Flex Software captures images of the ROI calibration plate at each instrument filter An ROI calibration passes if the collected image for each filter shows all wells of the ROI plate or array card Each well in the image must be distinct and visible at the same luminosity relative to the other wells in the image You can review the ROI calibration image for each filter set by selecting the desired filter combination from the Filter Set menu of the ROI tab in the Instrument Manager Status Image Passing image Green circles appear around all wells indicating that the wells calibrated successfully Each green circle indicates that the region of interest for the well position is sufficiently bright
174. e Safety Data Sheets SDSs before handling ATTENTION Produits chimiques dangereux Lire les fiches techniques de s ret de mat riels avant toute manipulation de produits CAUTION Hazardous waste Refer to SDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches techniques de s ret de mat riels et la r gulation locale associ es a la manipulation et l limination des d chets AN WARNING Hot lamp AVERTISSEMENT Lampe br lante WARNING Hot Do not remove lamp until 15 min after disconnecting supply AVERTISSEMENT Lampe br lante apr s avoir d connect le c ble d alimentation de Uappareil attendre environ 15 minutes avant d effectuer un remplacement de la lampe WARNING Hot Replace lamp with an Life Technologies lamp AVERTISSEMENT Composants br lants Remplacer la lampe par une lampe Life Technologies CAUTION Hot surface ATTENTION Surface br lante DANGER High voltage DANGER Haute tension WARNING To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Life Technologies qualified service personnel AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont ouverture n cessite l utilisation d outils Linstrument ne contient aucune pi ce r parable par Uutilisateur Toute interventi
175. e block and cause an abnormally high background signal Correct Incorrect Liquid is at bottom of well e Not centrifuged with enough force or s Not centrifuged for enough time 46 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Background calibration Performing the 1 In the Home screen of the QuantStudio 12K Flex Software click Instrument calibration Console 2 In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it 3 After the QuantStudio 12K Flex Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenance then click Background b In the Background Calibration screen click Start Calibration 5 Click Next then perform the calibration as instructed When the side door opens load the background plate or array card Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the barcode facing the front of the instrument IMP
176. e coordinate of the well that contains the lowest concentration of dye and that is encircled by a ring This well contains the optimal concentration of the custom dye at the given filter c Repeat steps a and b for the remaining filters d After you determine the optimum concentration for each filter determine the optimum concentration for the custom dye Compare the results from all filters then select the concentration that yields the highest possible signal in all filters 4 Discard the plate WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing 5 Inthe ROI dialog box click Finish to complete the calibration then click No when prompted to save the results Creating a custom IMPORTANT Wear powder free gloves while creating the dye plate dye plate 1 Prepare 2 mL of the custom dye at the concentration determined in Determining optimum dye concentration on page 184 2 Pipet 20 uL of the diluted custom dye to all wells of an optical reaction plate 3 Seal the wells of the reaction plate using an optical adhesive cover 4 Centrifuge the plate for 2 minutes at lt 1500 rpm Note The custom dye calibration plate must be well mixed and centrifuged 5 Verify that the liquid in each well of the plate is at the bottom of the well If not centrifuge the plate again at a higher rpm and for a longer period of time
177. e heated cover handle the assemblies as shown below After you remove each assembly from the QuantStudio 12K Flex Instrument place them on a clean dry surface or in its shipping container 1 Power off the QuantStudio 12K Flex Instrument and computer 2 When the QuantStudio 12K Flex System and computer are powered off disconnect all QuantStudio 12K Flex System components and package the cabling for the move 3 Prepare the QuantStudio 12K Flex Instrument for the move a Open the QuantStudio 12K Flex System access door b Firmly press down on the sample block handle pull the sample block from the QuantStudio 12K Flex Instrument then place it on a clean dry surface c Pinch the handle of the heated cover together then pull the assembly from the QuantStudio 12K Flex Instrument and place it on a clean dry surface Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 179 Powering On or Off Storing and Moving the System Moving the QuantStudio 12K Flex System Moving the QuantStudio 12K Flex System Reinstalling the QuantStudio 12K Flex System d Package the sample block and heated cover assemblies in a clean dust free container for the move Move the QuantStudio 12K Flex System according to the following guidelines Verify that the surface on which you will place the QuantStudio 12K Flex System can support at least 77
178. e library contain the sample name and the sample color The samples in the library may also contain comments about the sample In genotyping experiments the combination of the sample to test and the SNP assay to perform in one PCR reaction Each PCR reaction can contain only one sample and one SNP assay In quantification experiments the combination of the sample to test and the target to detect and quantify in one PCR reaction Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide security auditing and eSignature serial factor slope SNP SNP assay SNP Assay Library stage standard Glossary An optional software module that provides e System Security Controls user access to the software Provides a default Administrator user account You can define additional user accounts and permissions e Auditing Tracks changes made to library items actions performed by users and changes to the Security and Audit settings The software automatically audits some actions silently You can select other items for auditing and specify the audit mode Provides reports for audited library items Security and Audit changes and actions s Electronic Signature eSignature Controls whether users are permitted prompted or required to provide a user name and password when accessing certain software features You can select which features are controlled and the number of sig
179. e license key 1 Save the license lic file to the computer that contains the QuantStudio 12K Flex Software 2 In the main menu of the QuantStudio 12K Flex Software select Tools gt License Central 3 In the License Central dialog box click Install License 4 In the Open dialog box navigate to and select the license file then click Open 5 Click OK to close the License Central dialog box Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 4 Maintenance 4 Replacing the sample block Replacing the sample block Replace the sample block in the event of a hardware failure or to change the consumable format of the QuantStudio 12K Flex Instrument WARNING PHYSICAL INJURY HAZARD During instrument operation the sample block and heated cover can be heated to 100 C Before removing the sample block be sure to wait until it reaches room temperature Required materials e Safety glasses e Powder free gloves e Sample block Handling the To prevent damaging or contaminating the sample sample block block handle the assembly as shown below After the assembly has been removed from the QuantStudio 12K Flex Instrument place the sample block on a clean dry surface or in its shipping container Replacing the IMPORTANT If you are installing a sample block of a different format for example 96 sample block 384 well plate to array card you must al
180. e name of the target assay added to the well 100 character string Task The task assigned to the target in the well UNKNOWN NTC or STANDARD Reporter The reporter dye that labels the probe for the target assay 100 character string Quencher The quencher dye that labels the probe for the target assay 100 character string CT The calculated threshold cycle Cy for the target at the specified well Float Ct Mean The average Cy of the replicate wells for the specified target sample combination Float Ct SD The standard deviation of the average Cy of the replicate wells for the specified target Float Quantity e Unknown wells The calculated quantity for the sample at the well e Standard wells The quantity assigned to the standard at the well Float Quantity Mean e Unknown wells The average quantity of the replicate wells for the target sample e Standard wells The quantity assigned to the replicate wells for the target sample Float Quantity SD The standard deviation of the average quantity of the replicate wells for the target sample combination Float Automatic Ct Threshold Whether the threshold was determined automatically true or manually false true Or false Ct Threshold The threshold cycle C for the sample at the well Float Automatic Ct Whether the baseline was determined true Or false
181. e reaction plate or sample block e Fluorescent contaminant in the reaction decontaminate the sample block 2 Contact Life Technologies to order a replacement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Life Technologies for further assistance B Bunooysayqnos s4901g ajdwes pieg Aewy pue 3 eld N M MNW Bunesqneg olpnisjueng swaj stsoig panddy wl aping Jasf volessiulwpy pue adueuajulew W IS S Yd awl jeay Xa4 YZ 6L Problem symptom Possible cause Action Well displays the HIGHSD flag indicating that the C standard deviation for the replicate group exceeds the current flag setting e Droplets on the sides of the wells e Improper sealing or seal leaks e Condensation on the reaction plate e Inconsistent volumes across the plate Well displays the NOAMP flag indicating that the sample did not e Missing template e Excitation source in the If a well is flagged confirm the results 1 Select the flagged wells and the associated replication group s in the plate layout or well table View the amplification plot Rp vs Cycle and review the data for abnormalities 3 Hold the plate or array card up to a light source and check for condensation or evaporation If there are differences in fluid volumes check the heat seal of the wells with lower volumes for signs of damage or evaporation amplify R 4
182. e standard quantity for each point in a standard curve is calculated based on the starting quantity and serial factor In genotyping and presence absence experiments a DNA sample with a known genotype homozygous or heterozygous In the software the task for the SNP assay in wells that contain a sample with a known genotype In genotyping and presence absence experiments the part of the instrument run that occurs after amplification In genotyping experiments fluorescence data collected during the post PCR read are displayed in the allelic discrimination plot and used to make allele calls In presence absence experiments fluorescence data collected during the post PCR read are displayed in the presence absence plot and used to make detection calls Also called endpoint read In genotyping and presence absence experiments the part of the instrument run that occurs before amplification The pre PCR read is optional but recommended Fluorescence data collected during the pre PCR read can be used to normalize fluorescence data collected during the post PCR read PCR reaction component that contains the forward primer and reverse primer designed to amplify the target PCR reaction component that contains the primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target Fluorescent compound used to calibrate the instrument See system dye The fractional PCR cycle used for quantification accor
183. e tc Alte See E Aah eae Be ee Te A B oe de bes 53 When to perform the dye calibration cece eee eee eens 53 About the dye calibration 000 c eee eee eee ee eee ees 53 Preparing the calibration plate or array Card 00 c cee eee eee eee eee 55 Preparing the calibration plates n 000 eee eee eee eee 55 Performing the calibration ccc eee eee eee e eens 56 Normalization calibrationiis icgde0 X ZC E 49T E rda Ooz RA pie RAR Peed i ed 59 When to perform the calibration nsanu rnnr rnnr rnnr 59 About the normalization calibration data 59 Preparing the calibration plate or array card 00 0c nrnna nnna 59 Preparing the normalization plates 0 0000 e eee eee enter aes 59 Performing the calibration 27 wescd 4 csate ead eee ead ee de ed 60 Verifying the instrument performance cece eee eee eee e eens 63 When to perform the RNase P experiment 00 0000 c cece cece ee eee eee aee 63 About the RNase P kits heni nents do ore doe ei Pd eed 63 About the analysis 0000 cee eee eee eee e eee e eee eaes 64 Installation specification Swe a ele ite eee ad 65 Preparing the verification consumable cece cece e eee e eee e eee eeee 65 Preparing the TaqMan RNase P Instrument Verification Pate aaaaaaaaaanaan 65 Preparing an array card for instrument verification cece e ees 66 Running the experiment 00000 cee eee eee eee eee ees 68 Applied Biosystems QuantStudio 12K Flex Real Time PCR
184. eal Time PCR System Maintenance and Administration User Guide Documentation and Support Related documentation The following related documents are provided with the system Document Part no Description Applied Biosystems 4470050 Contains five individual booklets that explain how to perform the six different QuantStudio 12K Flex experiments on the QuantStudio 12K Flex Instrument Real Time PCR System The experiments include Standard Curve Relative Standard Curve and Multi well Plates and Array c lt omparative C7 Genotyping Presence Absence and Melt Curve Each Getting Card Experiments User Guide Started Guide booklet functions as both e A tutorial using example experiment data provided with the QuantStudio 12K Flex Software e A guide for your own experiments Intended for laboratory staff and principal investigators who perform experiments using the QuantStudio 12K Flex System Applied Biosystems 4470688 Explains how to install and maintain the QuantStudio 12K Flex Instrument nTM SaO ies Intended for laboratory staff responsible for the use and maintenance of the Real Time PCR System l l QuantStudio 12K Flex Instrument Quick Reference Guide Applied Biosystems 4470654 Explains how to prepare your site to receive and install the QuantStudio 12K QuantStudio 12K Flex Flex Instrument o es C Intended for personnel who schedule manage and perform the tasks required eg renee SEAS
185. eal time PCR the magnitude of normalized fluorescence signal generated by the reporter at each cycle during the PCR amplification In the ARn vs Cycle amplification plot ARn is calculated at each cycle as ARn cycle Rn cycle Rn baseline where Rn normalized reporter In genotyping experiments and presence absence experiments the difference in normalized fluorescence signal generated by the reporter between the pre PCR read and the post PCR read In the allelic discrimination plot genotyping experiments and the presence absence plot presence absence experiments ARn is calculated as ARn Rn post PCR read Rn pre PCR read where Rn normalized reporter See also normalized reporter Rn Expression estimation algorithm Cy that subtracts a baseline component and sets a fluorescent threshold in the exponential region for gene quantification Reactions that contain identical components and volumes but evaluate separate samples of the same biological source for example samples from three different mice of the same strain or separate extractions of the same cell line or tissue sample When an experiments uses biological replicate groups in a gene expression study the values displayed in the Biological Replicates tab are calculated by combining the results of the separate biological samples and treating this collection as a single population that is as one sample For AC computations normalizing by the endogen
186. eating the background plate background plate Remove an Applied Biosystems 96 384 Well Optical Reaction Plate from its box and place it on a clean dry surface Aliquot 20 uL deionized water to each well of the reaction plate Seal the plate using an optical adhesive cover or optical flat caps Use the plate for background calibration as you would a background plate from the spectral calibration kit 182 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix C Calibration Consumable Preparation Creating a background plate or array card Creating a 1 Remove an array card from its box and place it on a clean dry surface background array d 2 Using a permanent marker write Background on the side of the empty card car 3 Pipet 100 uL of deionized water into each of the eight reservoirs in the card a Place the array card on a lab bench with the foil side down b Load 100 uL of the solution into a pipette c Hold the pipette in an angled position 45 and place the tip into the fill port There is a fill port on the left arm of each fill reservoir the larger of the two holes Fill Vent A port port 1 2 3 4 5 6 GY ST IOT TOF OF OTT d Dispense the fluid so that it sweeps in and around the fill reservoir toward the vent port When pipetting the reagents into the array card pipet the entire 100 uL volume into the fill reservoi
187. eature you must similarly enable or disable the QuantStudio 12K Flex Software security see page 139 The QuantStudio 12K Flex Software cannot connect to QuantStudio 12K Flex Instruments that do not match security settings Note Secure mode limits the number of feature that are available from the QuantStudio 12K Flex Instrument touchscreen it does not provide user authentication functionality through the instrument touchscreen Enabling or disabling security 1 Ifthe instrument is in standby touch the QuantStudio 12K Flex System touchscreen to activate it then press 0 2 In the Main Menu touch Settings then touch Set Administrator Options 3 In the Set Administrator Options screen touch Secure Environment to enable checked or disable unchecked system security 4 Optional To change the administrator password a Touch Change Password b Enter the current password then touch Done c Enter the new password then touch Done d Reenter the password when prompted e Touch OK when prompted Note The default password for the QuantStudio 12K Flex Instrument touchscreen is password however the password can be changed during installation 5 Touch gm Save 6 Touch the Administrator Password field enter the administrator password then touch Done 7 Touch B to return to the Main Menu 172 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User
188. ed correctly Plug in and power on the QuantStudio 12K Flex System In the Home screen of the QuantStudio 12K Flex Software click Instrument Console In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments then review the Block Type field in the Instrument Properties pane The installation is successful if the QuantStudio 12K Flex Instrument powers on and if the Block Type field displays the correct type of sample block Note The Block Type field displays the type of sample block installed to the QuantStudio 12K Flex Instrument Perform the following calibrations in the specified order ROI calibration a b Background calibration 9 Uniformity calibration d Dye calibration e Normalization calibration Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 4 Maintenance 4 Replacing the heated cover Replacing the heated cover Replace the heated cover in the event of a hardware failure or if you want to change the consumable format of the QuantStudio 12K Flex Instrument WARNING PHYSICAL INJURY HAZARD During instrument operation the sample block and heated cover can be heated to 100 C Before removing the heated cover be sure to wait until it reaches room temperature Required materials e Safety glasses e Powder free gloves e Heated cover Handling the To prevent damaging or con
189. ed fluorescence data AR calculated by the QuantStudio 12K Flex Software The section begins with a column header followed by the amplification data where each row contains the data for a single well separated by tab characters If a well contains more than one assay target the QuantStudio 12K Flex Software lists the data for each additional assay on separate rows repeating the well number and sample information Column Description Output Well The number of the well on the consumable Integer 1 96 384 Cycle The cycle of the run during which the Integer QuantStudio 12K Flex Instrument recorded the fluorescence Target Name Genotyping experiments The name of the SNP lt SNP assay name gt assay assigned to the well and the allele name lt allele name gt All other experiments The name of the target Name of the target assigned to the well Rn The raw fluorescence for the associated well Float normalized to the fluorescence of the passive reference dye reporter signal or passive reference signal Delta Rn The baseline compensated R value for the Float associated well Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom 214 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix E File Format Reference E QuantStudio12KFlex export format Multicomponent T
190. eft hand table e Click Export Details to export the records in the left hand table and the associated details 5 Select a location for the export file enter a name for the file then click Save 6 Click OK in the confirmation message Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 149 Security Audit and Electronic Signature Managing electronic signature Printing audit records 1 2 3 4 Open an experiment eds or template edt file In the open experiment or template click kL gt Audit Click View Report to open the Print Preview dialog box Preview save or print the report e Click Save to save the report as a pdf or html file Enter the file name select a location select the file type then click Save e Click Print to send the report to the printer In the Print dialog box select the printer and print options then click OK Click 3 to close the Print Preview dialog box Managing electronic signature Enabling disabling IMPORTANT If you disable security you inactivate audit and electronic signature electronic functions No audit record is generated for the disabling of audit and electronic signature signature functions when you disable security 1 In the QuantStudio 12K Flex Software select Tools Security gt Settings 2 In the Security Settings dialog box select the e
191. elete a shortcut touch the shortcut of interest then touch if Remove Shortcut or touch Remove All to delete all shortcuts 3 When you are finished configuring the shortcuts touch M to return to the Main Menu Reviewing the You can use the QuantStudio 12K Flex Instrument touchscreen to view usage instrument statistics on the heated cover LEDs and other system components statistics Viewing the statistics 1 If the instrument is in standby touch the QuantStudio 12K Flex System touchscreen to activate it then press G 2 Inthe Main Menu touch Tools then touch Show Statistics 3 When you are finished touch EY to return to the Main Menu Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 171 A Manual Instrument Operation Administering the instrument from the touchscreen Enabling disabling instrument security The QuantStudio 12K Flex Instrument features a secure mode that can be enabled to restrict local instrument functionality When security is enabled use of the touchscreen is restricted to administrative functions that change the instrument settings After the QuantStudio 12K Flex Instrument is secured you must enter an administrator password to modify the instrument settings use the firmware tools or deactivate the secure mode IMPORTANT If you enable or disable the QuantStudio 12K Flex Instrument security auditing and electronic signature f
192. em you can save the results files to a USB drive and email them to Life Technologies technical support for a diagnosis Note We recommend running the self test as part of regular maintenance to ensure optimal performance of the QuantStudio 12K Flex Instrument 1 5 If the instrument is in standby touch the QuantStudio 12K Flex System touchscreen to activate it then press G In the Main Menu touch Tools then touch Run Self Test In the Self Test screen touch Kil Start Self Test then wait for the test to complete Optional When the QuantStudio 12K Flex Instrument completes the self test save the results to a USB drive a Plug a USB drive into the USB port on the right side of the QuantStudio 12K Flex Instrument touchscreen b Touch Save IMPORTANT Do not remove the USB drive from the QuantStudio 12K Flex Instrument until instructed to do so c When the QuantStudio 12K Flex Instrument finishes writing the results to the USB drive touch OK then remove the USB drive Touch Q to return to the Main Menu Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 165 Manual Instrument Operation Maintaining the instrument from the touchscreen Updating the instrument firmware 166 You can download QuantStudio 12K Flex Instrument firmware updates directly from the service section of the Life Technologies website After obtaining a
193. em 175 Placing the QuantStudio 12K Flex System on standby 000 cece eee eee ences 176 Powering on the QuantStudio 12K Flex System 000 ccc cece eee e eee e eee ens 176 Powering off the QuantStudio 12K Flex System 000 0c eee c eee eee eee eens 177 Storing the QuantStudio 12K Flex System 200 c cece eee rnrn rrr 178 RequiredimatetialS octies renie esi ee ga ny eee Rd RR ede fe Saeed R Fee eee eee 178 Preparing the QuantStudio 12K Flex Instrument 0 0000 e eee eee eee ee 178 Moving the QuantStudio 12K Flex System 2 0 0 0 ccc cece cence eee eee e eens 179 Requiredimaterials 2 c 2 fic ZE aan T Que i hi aot Z elo lee Scola colt cath L 179 Handling the sample block and heated cover cece cece cence eee aee 179 Preparing the QuantStudio 12K Flex System components 00 00eeeee eens 179 Moving the QuantStudio 12K Flex System nn nannu 000 eee ee rnrn rrn 180 Reinstalling the QuantStudio 12K Flex System 0c cece eee eee eee aes 180 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Contents APPENDIX C Calibration Consumable Preparation 181 Creating a background plate or array card 0 ccc eee aes 182 Required materials 000 ccc cect ttt eee eee ee eens 182 Creating a background plate nunnan aenaran ernearen 182 Creating a background array Card
194. emarks mentioned herein are the property of Life Technologies Corporation or their respective owners TaqMan is a registered trademark of Roche Molecular Systems Inc Twister is a registered trademark of Caliper Life Sciences Apple Safari and Macintosh are registered trademarks of Apple Inc Microsoft Windows Excel and Internet Explorer are registered trademarks of Microsoft Corporation Mozilla and Firefox are registered trademarks of Mozilla Foundation Adobe Acrobat and Reader are registered trademarks of Adobe Systems Inc iLink is a registered trademark of Zymark Corporation 2012 Life Technologies Corporation All rights reserved Contents About This e Hl LT 12 S Tl sts TTT 13 UCI NCES a R ot Ad ay caida AAE dE 13 ASSUMPTIONS A aol ena ald ee a e AE A ee a eit ee iN oo Meath a tor le utara 13 Safety informati n s pe desiren eee ee Salen ed dale ayaa a a eaten se eee Aa R ak 14 Safety alert words lt sve rr dN ved eee ade decide Ride vie peed ae oe he N oe eee ee oes 14 Safety data sheets SDSS nits cccse tec eed tgs ed bal coed ae ah dae eh 14 Safety labels on instruments 000 cece cece rnanan 15 Using this quide arira eee rahe seeds Gabe hehe Be Sot Pee Seed ee ee eee ad 16 CHAPTER 1 Getting Started e 17 About the QuantStudio 12K Flex System 2 0c cece cece rnnr rnnr rrer 18 About data colection gesce beste aha TE eae EE Neg a Meare dea 18 Instrument filters and supported dyes cece raer 19 Spec
195. ements for safety Low Voltage Directive 2006 95 EC This instrument has been tested to and complies with standards EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements EN 61010 2 010 2003 Particular Requirements for Laboratory Equipment for the Heating of Materials EN 61010 2 081 2002 A1 2003 Particular Requirements for Automatic and Semi Automatic Laboratory Equipment for Analysis and Other Purposes The Applied Biosystems QuantStudio 12K Flex Real Time PCR Instrument meets European requirements for emission and immunity EMC Directive 2004 108 EC EN 61326 1 2006 Electrical equipment for measurement control and laboratory use Part 1 General EMC requirements Group 1 Class B Australia and New Zealand EMC standards This instrument has been tested to and complies with standard AS NZS 2064 Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment 244 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chemical safety Appendix G Safety G Chemical safety General chemical Chemical hazard warning safety WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precaut
196. enArray Plate Instrument Verification Reagents Kit into the Sample Plate field Insert the 384 well sample plate with the foil cover still in place Press on the plate until it snaps into place Note Do not remove the foil from the 384 well sample plate at this stage Enter the data for the OpenArray plate a Select 1 from the Samples Per Subarray drop down list b In the plate holder Position 1 text field enter RNase P the sample loaded into first position of the plate holder c Place a thawed OpenArray plate into the plate holder When handling the OpenArray plate e Always hold the OpenArray case by the edges e If you drop a loaded OpenArray plate discard it in the sharps waste container s Load the OpenArray plate within an hour after you open it Hold the OpenArray case by the edges and place it in the plate holder with the barcode facing up and to the left Click Next Verify that the Tip Status window in the software matches the state of the tips on the deck Ensure that e Gray areas in the Tip Status window indicate that tips are not present e White areas indicate that tips are present If the software and the tips on the deck do not match click the appropriate section in the Tip Status window Select the state of the tip boxes by clicking on the sections Tip Box 1 Tip Box 2 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration U
197. ep then touch Add e Adda melt curve to the end of the thermal profile touch K 9 Add Melt Curve e Change the time or temperature of a stage or step touch the time temperature field of the stage or step modify the settings as desired then touch Close e Change the cycle parameter of a stage touch the cycle field modify the setting as desired then touch Close e Delete a stage or step from the thermal profile touch the stage or step you want to remove then touch i Delete 7 When finished modifying the parameters touch 6 Save 8 In the Save Experiment screen touch each field to set the experiment name reaction volume barcode and any additional information to save to the experiment 9 When finished touch Save amp Start Run to start the experiment 10 In the Start Run screen touch each field as needed to modify the associated parameter then touch Start Run Now to start the experiment Note When the run is complete touch ea to unload the plate You can download the experiment results to a computer if the QuantStudio 12K Flex Instrument is connected to a network or you can copy the data to a USB device see Transferring experiments templates and results data on page 161 160 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Transferring You can transfer experiments templates and results data experiments to from the QuantStudio 12
198. eplacement RNase P OpenArray plate fails contact Life Technologies for further assistance Through hole displays the HIGHSD flag the C standard deviation for the e Droplets on the sides of the through holes If a through hole is flagged confirm the results S 1 Select the flagged through holels and the associated replication group s Hae ia exceeds the current e Improper sealing or seal leaks in the plate layout or through hole table ag setting J a e Condensation on the reaction plate 2 View the amplification plot R vs Cycle and review the data for e Inconsistent volumes across the abnormalities plate 3 Hold the OpenArray plate up to a light source and check for leaks and Through hole displays the NOAMP flag lt Missing template bubbles the sample did not amplify e Excitation source in the 4 Check the ROX image files for non uniformity as explained in Viewing the instrument stopped functioning ROX image files on page 108 Non uniformity can indicate problems with Through hole displays the NOISE flag Droplets on the sides of the plate loading the through hole produced more through holes 5 Order and perform a replacement RNase P experiment Properly seal and noise in the amplification plot than e Improper sealing or seal leaks centrifuge the RNase P plate If the replacement RNase P plate fails other through holes on the plate l contact Life Technologies for further assistance e C
199. er flathead Power off then unplug the QuantStudio 12K Flex Instrument Allow it to cool for 15 minutes Using a flat head screwdriver unscrew and remove the fuse holder ML Remove each fuse from its fuse holder and inspect it for damage Carbon typically coats the inside of failed fuses Good Failed Replace each failed fuse with a 12 5A Time Lag T 250VAC 5 x 20 mm Fuse Note The voltage and amperage ratings are on the fuse holder Install the fuse holder Plug in then power on the QuantStudio 12K Flex Instrument The installation is successful if the instrument powers on Note Fuse failure can result from fluctuations in the supplied power to the QuantStudio 12K Flex Instrument To prevent further failures consider installing an electrical protective device such as a UPS or a surge protector 114 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 4 Maintenance 4 Updating the Windows operating system Updating the Windows operating system Do not upgrade or update the Microsoft Windows operating system of the computer running the QuantStudio 12K Flex Software without first consulting the software release notes or the Life Technologies website Future versions and updates to the Windows operating system can conflict with the QuantStudio 12K Flex Software To determine compatibility of an upgrade or update 1 Open C
200. er to rinse wells after cleaning with bleach or ethanol solution If the contamination remains repeat steps 2 through 5 then clean the contaminated wells of the sample block using 10 bleach solution a Pipet a small volume of 10 bleach solution into each contaminated well b In each contaminated well pipet the solution up and down several times to rinse the well If you are decontaminating an array card or OpenArray plate sample block swab the surface of the block that contacts the consumable c Pipet the bleach solution to a waste beaker Repeat steps 5 through 9 to rinse the wells of the sample block and to verify that you have eliminated the contamination IMPORTANT Always use deionized water to rinse wells after cleaning with bleach or ethanol solution If the contamination remains contact Life Technologies Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 113 4 Maintenance Replacing the instrument fuses Replacing the instrument fuses Required materials Replacing the fuses Replace the QuantStudio 12K Flex System fuses when the fuses fail CAUTION FIRE HAZARD For continued protection against the risk of fire replace fuses only with listed and certified fuses of the same type and rating as those currently in the QuantStudio 12K Flex Instrument Fuses 12 5A Time Lag T 250VAC 5 x 20 mm 2 Safety glasses Powder free gloves Screwdriv
201. erate the Twister Robot By default the software is installed to the TM C drive of the QuantStudio 12K Flex System computer and it consists of the components shown below e QuantStudio 12K Flex Instrument Control Program ICP Calibrates the Twister Robot and stores all taught positions e QuantStudio Adapter Driver for iLink PRO Software Coordinates the operation of the Twister Robot and QuantStudio 12K Flex Instrument e Microsoft software Provides the Microsoft services used by the Twister Robot Software The components include Microsoft Data Access Components MDAC Microsoft NET Framework Microsoft SQL 2005 Manager and Microsoft VBA Service Packs e Automation Controller Software and iLink PRO Software Software and automation controller software applications that can be used to automate the operation of the Twister Robot and the QuantStudio 12K Flex Instrument Note The iLink PRO Storage software for the Twister Robot racks is used with the iLink PRO automation control software to set up the initial material layout Before you install third party software to the computer running the QuantStudio 12K Flex Software confirm that the software will not e Restrict Ethernet communication e Interfere with QuantStudio 12K Flex Software operation see below To confirm that third party software does not interfere with the QuantStudio 12K Flex Software 1 Instal
202. ermissions Audit Reason for Delete Create reason for change Change Update Delete or modify reason for change Event Manager Update Update the event manager E signature Manager Update Enable or disable e signature E signature Type Create Create an e signature meaning Delete Delete an e signature meaning Update Edit an e signature meaning or an e signature action E signature Function Update Edit an action requiring e signature T Creates one role assignment record for each permission in a role Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 147 6 Security Audit and Electronic Signature Generating audit reports Reviewing the action log The Action Record log lists system specified audit events All items in the action log are audited silently except for the items noted as configurable Configurable items may include comments in the action log Audit Settings Update Auditing Event Archive Restore Purge Configuration Import Export Data Audit Archive Restore Purge Login Success Failure Logout Success Run Start Stop Completed Failed Aborted Error User Account Create Update Viewing and printing audit reports 1 2 3 6 Select the System Configuration History tab Display the records of interest Filter the list to decrease the time required to generate reports IMPORTANT You
203. ers field select the folder that contains the files that you want to download b In the Experiments field select the files to download To select multiple files Ctrl click or Shift click files in the list c When you have selected the files that you want to download click Download d In the Send experiment to instrument dialog box select the folder to which you want to download the selected file s then click Open Note You can also use the Folders and Experiments fields to s Create or remove directories on the QuantStudio 12K Flex Instrument s Add delete or download experiments on the QuantStudio 12K Flex Instrument Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 133 5 Networking Monitoring a QuantStudio 12K Flex Instrument Enabling or changing the calibration reminders The calibration reminders settings allow you to configure the QuantStudio 12K Flex Software to alert you by email when the QuantStudio 12K Flex Instrument requires calibration The notifications settings feature is optional and it does not affect performance IMPORTANT The QuantStudio 12K Flex Software transmits email only while the QuantStudio 12K Flex Instrument is monitored If the network connection is interrupted the software stops transmitting updates Required information The QuantStudio 12K Flex Software requires access to a Simple Mail Transfer P
204. ervice DHCP Touch Obtain an IP address automatically then touch Save Static IP 1 Touch Use the following IP address 2 Touch the IP Address field enter the IP address using the keypad then touch Done 3 Repeat step 2 to assign the e P addresses for the DNS Servers primary and secondary e Subnet Mask setting e Default Gateway setting 4 Touch S Save to save the settings then touch OK when prompted 6 Touch Te to return to the Main Menu 170 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix A Manual Instrument Operation Administering the instrument from the touchscreen Defining the You can use the QuantStudio 12K Flex Instrument touchscreen to map the shortcut system shortcuts buttons that appear in the Main Menu You can configure shortcuts to automatically open specific files and folders so that you can access data quickly and easily without having to navigate to it Defining the shortcuts 1 If the instrument is in standby touch the QuantStudio 12K Flex System touchscreen to activate it then press 0 2 In the Main Menu configure the shortcuts as desired To add a shortcut a Touch the shortcut of interest then touch qe Set Shortcut b Touch From Templates to link to a specific template file or touch From Folders to link to a folder c Touch the desired template file or folder to configure the shortcut To d
205. es not eject the plate remove the plate according to Table 4 Troubleshooting uniformity calibrations on page 74 8 Discard or store the plate or array card Consumable Action Array card Discard the array card Plate Return the ROI calibration plate to its packaging sleeve then return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate is stored in its original packaging sleeve you can use it to calibrate a QuantStudio 12K Flex System 3 times for up to 6 months after you open it 9 Inthe Uniformity Calibration screen click Finish to complete the calibration then click Yes when prompted to save the results 52 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Dye calibration When to perform the dye calibration About the dye calibration Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Dye calibration During a dye calibration the Applied Biosystems QuantStudio 12K Flex Real Time PCR System e Collects spectral data from a series of dye standards e Stores the spectral information for the dye standards in a dye calibration file The QuantStudio 12K Flex Software uses the pure spectra data during experiment runs to characterize and distinguish the individual contribution of each dye in the total fluorescence collected by the QuantStudio 12K Flex Ins
206. es website To view the demonstration go to www2 appliedbiosystems com lib multimedia taqman_tlda tlda_1 cfm 1 Remove the tubes of calibration solutions from the freezer allow them to thaw then vortex the tubes to mix the contents well 2 Remove the array cards from their box and place them on a clean dry surface 3 Mark the side of the empty array cards with e Background s FAM e ROI s ROX e VIC e FAM ROX s VIC ROX 4 For each array card pipet 100 uL of the appropriate calibration solution into each of the eight reservoirs in the array card a Place the array card on a lab bench with the foil side down b Load 100 uL of the calibration solution into a pipette Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 37 38 Place the filled array card s into a centrifuge array card Place the filled carrier clips into the centrifuge buckets Calibrating Multi Well Plate and Array Card Sample Blocks Preparing array cards for instrument calibration c Hold the pipette in an angled position 45 degrees and place the tip into the fill port There is a fill port on the left arm of each fill reservoir the larger of the two holes Fill Vent A port port 1 2 3 4 5 6 Ql fO f O O f Ql l IG i d Dispense the fluid so that it sweeps in and around the fill reservoir toward the vent port When pipetting the reagents into the array
207. escribes how to configure the security audit and electronic signature functions of the QuantStudio 12K Flex Software Appendix A Manual Instrument Operation Describes how to operate the QuantStudio 12K Flex Instrument manually using the touchscreen interface Appendix B Powering On or Off Storing and Moving the System Describes how to store move and reinstall the components of the system Appendix C Calibration Consumable Preparation Describes how to prepare array cards and OpenArray plates for calibration and verification of the QuantStudio 12K Flex Instrument The appendix also describes how to create a background plate or array card in the event that one is unavailable and how to create a dye plate or array card that can be used to calibrate the system for a dye not manufactured by Life Technologies Appendix D Command line Software Operation Describes how to use the QuantStudio 12K Flex Software command line application Appendix E File Format Reference Provides specifications for files that the QuantStudio 12K Flex Software imports exports and stores Appendix F Parts and Materials Describes how to order parts accessories and consumables for the QuantStudio 12K Flex System 16 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Getting Started This chapter covers m About the Qu
208. etwork In the configuration shown below computers on the network can exchange experiment data with the QuantStudio 12K Flex System computer however the QuantStudio 12K Flex Instrument can be neither monitored nor controlled remotely because it is physically isolated from the network DHCP server Networked Networked Instrument computer computer Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 127 5 Networking Network setup workflow Networking S guidelines and best practices Consult a network administrator We recommend that you consult a network administrator before connecting the QuantStudio 12K Flex System to your laboratory network To enable the full functionality of the QuantStudio 12K Flex Software the computer requires a network connection Limit remote monitoring to 10 computers Avoid using more than 10 computers to simultaneously monitor the QuantStudio 12K Flex Instrument remotely Although the QuantStudio 12K Flex System supports remote monitoring from multiple computers each connection taxes the instrument microprocessor Too many connections can overburden the QuantStudio 12K Flex System and result in instrument errors Note The effects of an overburdened QuantStudio 12K Flex System are evident in the Temperature Plot during a run Symptoms can include extended hold times or brief unexpected plateaus in the in
209. ety ic dubs s000s bass T RTE Saas daw sae Abie L 245 SDSS ih at xd nw ew nS ow eee Oe eee Rake eaT ase 246 Chemical waste safety 6 6 ccc nent eee 246 Biological hazard safety 0 0 6 ccc ccc eee een eens 248 Safety alerts sesso bss sed nad pea rabid Raed aG a pi aoaaa etude a Aa E EKETE 249 General alerts for all chemicals 066 nanena 249 General alerts for instrumentation 006 c cece nee eee 249 Specific alerts for instrumentation n cee eee eee 249 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 237 C Safety Instrumentation safety Instrumentation safety Symbols on Electrical symbols on instruments instruments The following table describes the electrical symbols that may be displayed on Life Technologies instruments Symbol Description Indicates the On position of the main power switch Indicates the Off position of the main power switch Indicates a standby switch by which the instrument is switched on to the Standby condition Hazardous voltage may be present if this switch is on standby Indicates a terminal that may be connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates a termin
210. ex System set for mDNS may not be visible to other nodes that are separated by a router hub or another network device s IPv4 link local IPV4LL in the RFC also known as Automatic Private IP Addressing APIPA or Internet Protocol Automatic Configuration IPAC Note When the QuantStudio 12K Flex System is set for DHCP APIPA is automatically enabled and the QuantStudio 12K Flex System provides an IP address when no address is supplied by the DHCP server 126 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Example network layouts Chapter 5 Networking 5 Networking overview Example 1 In the following example one or more QuantStudio 12K Flex Instruments which have been configured for dynamic host configuration protocol DHCP operation are connected to a network by their Ethernet ports In this layout any computer on the network can monitor or control the QuantStudio 12K Flex Instrument Experiments can be started remotely from the networked computer or locally from the QuantStudio 12K Flex Instrument touchscreen Note A networked computer running the QuantStudio 12K Flex Software can simultaneously control up to 4 instruments and monitor up to 15 instruments that have been connected to the network DHCP server Networked Networked computer instrument s Example 2 The QuantStudio 12K Flex System computer can be connected to the n
211. extracts the dye calibration data it evaluates the fluorescence signal generated by each OpenArray calibration case in terms of the collective spectra for the entire tool Dye spectra are generally acceptable if they peak within the same filter as their group but diverge slightly at other wavelengths The QuantStudio 12K Flex Software can compensate for some differences in a spectral profile by replacing the spectra of unacceptable wells with the spectra of other regions of the OpenArray Calibration Case auto repairing The QuantStudio 12K Flex Software allows only a few replacements and it may reject the calibration if the spectra between neighboring wells vary significantly e Wear gloves that are one size smaller than the size you typically wear to help prevent excess glove material from contacting the OpenArray Calibration Cases while loading e Hold OpenArray Calibration Cases by the edges e Ifyou drop a loaded OpenArray calibration case discard it in the appropriate waste container IMPORTANT Wear powder free gloves while preparing the OpenArray Calibration Cases 1 Load the empty OpenArray Calibration Cases into the OpenArray Calibration Carrier according to the labels on the cases a Remove the OpenArray Calibration Cases from their packaging b Remove the protective film from all of the OpenArray Calibration Cases Applied Biosystems QuantStudio 12K Flex Real Time PCR System Main
212. ey for the QuantStudio 12K Flex Software or an associated module or if your license file has expired you can use the License Central feature to activate the license as explained below IMPORTANT An internet connection a web browser and a valid email account are required to activate a QuantStudio 12K Flex Software license If the computer that contains the QuantStudio 12K Flex Software is not connected to the internet or it lacks a web browser application contact Life Technologies support to request the license file 1 In the main menu of the QuantStudio 12K Flex Software select Tools License Central 2 In the License Central dialog box select the license of interest from the table click Renew License then wait for the default web browser application to connect to the Life Technologies website 3 In the Life Technologies Software License Activation website click QuantStudio 12K Flex Software from the list of products then activate the license as instructed After you successfully activate the license the Life Technologies website emails you the activated license file lic for you to install on your computer Installing a license file After you activate your license and receive an activated license file lic install the file as explained below to unlock the QuantStudio 12K Flex Software or module Note Each license file is generated specifically for the computer that was used to activate th
213. f Test 97 system shortcuts instrument 171 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 277 Index T TAMRA dye 19 53 TaqMan OpenArray plate guidelines for handling 95 TaqMan reagents 267 TaqMan RNase P Fast 384 Well Instrument Verifica tion Plate 63 233 target 267 target color 267 Target Library 267 task 267 technical replicate 267 technician user role 143 Temperature Plot 267 temperature requirement 22 template 267 template file 267 third party software 31 threshold 268 threshold cycle 268 threshold cycle CT 268 timeout session 139 155 touchscreen instrument 24 158 training information on 252 transfer data to from instrument 133 162 tray arm 24 troubleshoot background calibration 73 dye calibration 75 instrument fuses 114 instrument verification 77 105 normalization calibration 76 optical calibration 73 RNase P instrument verification experiment 77 105 ROI calibration 72 sample block decontamination 111 uniformity calibration 74 U ultraviolet light safety 242 uniformity calibration 49 87 268 data 49 87 perform 51 troubleshoot 74 when to perform 49 87 uninterruptable power supply requirements 29 unknown 268 unknown IPC wells 268 update firmware 116 166 operating system 115 service packs 115 software 116 UPS requirements 29 USB drive transfer data 161 USB ports 24 25 161 user account activate suspended 142 create
214. f the moving parts hazard and have been adequately trained 7 After loading the plate or array card start the calibration a In the Dye Calibration screen select Check the box when the normalization calibration plate has been loaded then click Next b In the Run screen click START RUN to start the calibration IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature 8 When the run is complete and the QuantStudio 12K Flex Software displays the Analysis screen verify the status of the calibration The analysis status indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or the data it collected is unusable Analysis status Action Passed Enter any comments you have in the Comments field click Next then remove the plate or array card when the QuantStudio 12K Flex Instrument ejects the tray arm Failed Troubleshoot the failed calibration as described in Table 6 Troubleshooting normalization calibrations on page 76 WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing
215. formation that appears in the SDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following e Open To view the document e Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you choose Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Chemical waste hazards CAUTION HAZARDOUS WASTE Refer to Safety Data Sheets and local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Life Technologies instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles 246 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix G Safety G Chemical safety Chemical waste safety guidelines To minimize the hazards of chemical waste e Read and understand the Safety Data Sheets SDS
216. fying the instrument performance 5 Centrifuge and seal the array card as explained in steps 6 through 11 on page 38 6 Run the prepared array card as soon as possible after filling it Store the array card in a dark place until you are ready to run it IMPORTANT Do not expose the array card to light until you are ready to run it The fluorescent dyes in the array card are photosensitive Prolonged exposure to light can diminish the fluorescence of the dye Running the 1 In the Home screen of the QuantStudio 12K Flex Software click Instrument experiment Console 2 In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it 3 After the QuantStudio 12K Flex Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the RNase P wizard a Click Maintenance then click RNase P Run b In the RNase P Run screen click Start RNase P Run 5 Complete the calibration as instructed by the wizard When the side door opens load the RNase P plate or array card Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter s B Load both plates array cards and OpenArray plates wit
217. h the barcode facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 6 After loading the plate or array card start the calibration a In the Overview screen select Check the box when the RNase P calibration plate has been loaded then click Next b In the Run screen click START RUN to start the calibration IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature 68 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Verifying the instrument performance 7 When the run is complete and the QuantStudio 12K Flex Software displays the Analysis screen verify the status of the run Analysis status Action Passed The QuantStudio 12K Flex System passed the RNase P run Go to step 12 on page 70 Failed The QuantStudio 12K Flex System failed the RNase P run Go to step 8 to review the data for outliers If the run fails the QuantStudio 12K Flex Software may have included outliers that caused the initial analysis to fail Experimental error may c
218. hat is used to perform background and uniformity calibrations of the QuantStudio 12K Flex System with an OpenArray sample block The plaque consists of a thin sheet of black plastic that has two distinct sides shown below Black side Orange side s Dull matte black in color e Glossy dark orange in color e Completely smooth e Textured with a faint lattice pattern e Performs the background calibration e Performs the uniformity calibration Ca ring for the The OpenArray Calibration Plaque is sensitive to light and must be kept clean at all Open Array times Adhere to the following handling storage and cleaning guidelines when using Calibration Plaque Te tool Action Guidelines Handling When handling the OpenArray Calibration Plaque e Always wear powder free gloves e Grasp the tool by the edges e Ensure the tool does not become dirty or dusty Storing When not in use store the OpenArray Calibration Plaque e At room temperature e In the original packaging sleeve or in a clean plastic bag e Ina dark clean place such as a drawer or cabinet Cleaning If the OpenArray Calibration Plaque becomes dirty clean the tool as follows a Place the OpenArray Calibration Plaque on a clean dry surface b Pipet a small volume of 95 ethanol or 95 isopropanol solution onto a lint free wipe then thoroughly swab the surface of the tool c Use a lint free wipe to absorb the excess solution
219. he PCR run 259 Glossary negative control NC negative control blocked IPC wells negative control IPC wells no amplification control NAC no template control NTC nonfluorescent quencher minor groove binder NFQ MGB normalization calibration normalized quantity normalized quantity mean normalized quantity SE normalized reporter Rn omit well outlier 260 The task for targets or SNP assays in wells that contain water or buffer instead of sample No amplification of the target should occur in negative control wells Previously called no template control NTC In presence absence experiments wells that contain IPC blocking agent instead of sample in the PCR reaction No amplification should occur in negative control blocked IPC wells because the reaction contains no sample and amplification of the IPC is blocked Previously called no amplification control NAC In presence absence experiments wells that contain IPC template and buffer or water instead of sample Only the IPC template should amplify in negative control IPC wells because the reaction contains no sample Previously called IPC See negative control blocked IPC wells See negative control NC Molecules that are attached to the 3 end of TaqMan probes When the probe is intact the nonfluorescent quencher NFQ prevents the reporter dye from emitting fluorescence signal Because the NFQ does not fluoresce it produce
220. he QuantStudio 12K Flex Software can export the data used to generate the data multicomponent plot of a real time PCR experiment The multicomponent data tracks the raw fluorescence of all reporter dyes present on the reaction consumable throughout the duration of the experiment run The section begins with a column header followed by the multicomponent data where each row contains the data for a single well separated by tab characters The multicomponent data contains a dye column for each dye present on the reaction consumable including reporter dyes quencher dyes except non fluorescent dyes and the passive reference Column Description Output Well The number of the well on the consumable Integer 1 96 384 Cycle The cycle of the run during which the Integer QuantStudio 12K Flex Instrument recorded the fluorescence data lt Dye name gt The raw fluorescence for the designated dye Float measured by the QuantStudio 12K Flex Instrument at the specified well and cycle Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Results data The QuantStudio 12K Flex Software can export the results data from an analyzed experiment file The format and content of the results data depends on the experiment type and the analysis settings The section begins with a column header followed by the results data where each row contains the data for a single
221. he Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature Rotate the plaque When the instrument door opens and you are prompted to rotate the OpenArray Calibration Plaque 1 Rotate the OpenArray Calibration Plaque 180 then place it back into the plate retainer orange side up IMPORTANT Do not flip the OpenArray Calibration Plaque over The orange side of the tool must be facing up Click OK to close this dialog box then click START RUN in the Run screen to perform the second reading 88 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 3 Calibrating OpenArray Plate Sample Blocks Uniformity calibration Complete the IMPORTANT Wear powder free gloves and safety glasses when you handle the calibration OpenArray Calibration Plaque 1 Verify the status of the calibration The Analysis Status displayed by the QuantStudio 12K Flex Software indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or the data it collected is unusable Analysis statue Action
222. he case may interfere with thermal uniformity and can fluoresce Make sure you thoroughly clean each case IMPORTANT Run the prepared calibration OpenArray plates within one hour after loading them Discard the filled plate after a successful calibration 102 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 3 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance Running the 1 In the QuantStudio 12K Flex Software Home screen click Instrument Console experiment 2 In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it 3 After the QuantStudio 12K Flex Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the RNase P wizard a Click Maintenance then click RNase P Run b In the RNase P Run screen click Start RNase P Run 5 Complete the calibration as instructed by the wizard When the instrument door opens load the OpenArray plate into any position on the plate carrier Confirm that the OpenArray plate is positioned so that the barcode is closest to the A1 position on the plate retainer and that the plug is oriented toward the front of the instrument IMPORTANT P
223. he dye Note Among wells containing the same dye variations in spectral position and peak position are caused by minor differences in the optical properties and excitation energy between the individual wells 8 Verify the status of the calibration If the calibration Passed If all spectra are acceptable finish the calibration a Click Next b Enter any comments you have in the Comments field click Finish then click Yes when prompted to save the calibration results Failed Create another custom dye plate using the next dye concentration greater than the concentration determined in Determining optimum dye concentration on page 184 then perform the calibration again Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 187 Calibration Consumable Preparation Creating a custom dye plate for calibration 188 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Command line Software Operation This appendix covers OVET VIEW T ie rc tthe bon tee atria Soa te e Ed 190 Supporting files for experiment creation 0 eee eee 191 Precedence rules for experiment file generation 0 0 ccc eens 192 Running the command line application 6 0 c cece eee eee eee 193 Command syntax and arguments eee nee eee 194 Examples torei Ta RT Sha cock trate E E EEE E NA NA 9 take eae 197 Applied Biosystems Q
224. he instrument performance 5 Load the OpenArray case with OpenArray Immersion Fluid IMPORTANT Do not expose the Immersion fluid in the OpenArray cases to air for more than 60 minutes a Remove the sealed OpenArray case from the press grasping the case by the edges b Insert the syringe tip into the loading port at end of the sealed OpenArray case then dispense the fluid completely in one gentle continuous motion IMPORTANT Expel the OpenArray Immersion Fluid slowly If injected too quickly the fluid can flush out the samples suspended in the through holes Note Minimize creating air bubbles when you dispense the fluid one small air bubble is acceptable Ss Sealed OpenArray plate FILL label indicates the location of the fill port Fill port c While holding the OpenArray plate vertically seal the loading port with OpenArray Plug by inserting the plug into the port and twisting the plug clockwise until hand tight Once secure remove the knob from the plug 4 OpenArray plug insert and twist d Clean the case with a laboratory wipe that has been thoroughly sprayed with ethanol To dry the case wipe the case downward with a clean laboratory wipe Gently handle the case be sure to not apply pressure on the OpenArray plate within the case The sealed OpenArray plate can be loaded into the QuantStudio 12K Flex Instrument Note Dust or excess sample on t
225. he replacement RNase P plate or array card fails contact Life Technologies for further assistance sy20 g ajdues pueg Aewy pue aed N M MNW Bunewqneg Z 4aydeyg 6unooysayqnos E 84 oipn s ueng swas soig panddy WL aping J SN uoNejSiulWpy DUS aueUajuleW WaISkS Yd aw jeay Xa4 YZ Problem symptom Possible cause Action Well displays the BADROX flag indicating that the passive reference signal is unacceptable for the normalization of the reporter dye signal e Droplets on the sides of the wells e Improper sealing or seal leaks e Condensation on the reaction plate If a well is flagged confirm the results 1 Select the flagged well s in the plate layout or well table 2 View the amplification plot R vs Cycle and review the data in the C region for abnormalities Examine the reaction plate to check for condensation and or inconsistent reaction volumes Contact Life Technologies to order a replacement TaqMan RNase P plate or array card kit Repeat the experiment and make sure to properly seal and centrifuge the RNase P plate or array card If the replacement RNase P plate or array card fails contact Life Technologies for further assistance Well displays the BLFAIL flag indicating that the software cannot calculate the best fit baseline for the data e Amplification too late e No amplification Well displays the CTFAIL flag indicating that the software can
226. hen to perform 41 ROX dye 19 53 264 RS232 port 25 28 run method 264 runtype 18 run experiments 160 S safety Array Card Staker Sealer 37 bar code scanner 243 before operating the instrument 241 biological hazards 248 chemical 245 chemical waste 246 electrical 242 ergonomic 243 guidelines 245 246 247 instrument operation 241 laser 243 moving and lifting 180 241 moving parts 242 physical hazard 242 repetitive motion 243 standards 244 ultraviolet light 242 workstation 243 safety labels on instruments 15 240 safety standards 244 safety symbols on instruments 238 sample 264 sample block 24 decontamination 111 handling 111 119 installation 119 sample definition file 264 sample file 191 file format 206 Sample Library 264 sample SNP assay reaction 264 sample target reaction 264 scientist user role 143 SDSs about 14 description 246 obtaining 246 252 seal array cards 37 security administrator 138 enable disable 139 instrument setup 172 policies 139 software setup 139 security and auditing 265 276 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide security administrator account setup 139 disable effect on audit and e sig 139 enable disable 139 export settings 153 export user account settings 153 import settings 153 import user account settings 153 notification 139 140 overview 138 security policies 139 spaces in user names 140 user accounts 14
227. hout the aid of a quencher dye d Click Close 3 In the Dye window enter a temperature setting for the calibration Set the temperature to match the temperature at which you intend to collect data For example the temperature for all Life Technologies system dyes is 60 C because data collection for TaqMan reagents occurs during the 60 C extension step of the PCR 4 Load the appropriate dye plate into the plate adapter select Please check the box when the dye calibration plate has been loaded click Next twice then click START RUN to start the calibration 5 When the run is complete and the QuantStudio 12K Flex Instrument ejects the plate remove and discard the plate or array card WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing 6 In the Dye dialog box of the QuantStudio 12K Flex Software click Next 7 Verify the grouping of the dye spectra 186 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix C Calibration Consumable Preparation Creating a custom dye plate for calibration a In the plate layout select the wells of the plate b Inspect the raw data For each spectrum verify that the peak is e Within the detectable range for the QuantStudio 12K Flex System e Free of irregular spectral peaks e Present at the correct filter for t
228. ication stage and hold stage A sample that you dilute and amplify along with unknown samples This dilution series can contain known starting quantities of the target of interest absolute standard curve or it can be of known dilution factor relative standard curve Following the run the software interpolates the Cr values of the unknowns to this curve yielding either specific quantities of the target for absolute curves or relative quantities for relative dilution curves See also standard curve Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 265 Glossary standard curve standard curve method standard dilution series standard quantity starting quantity step SYBR Green reagents In standard curve and relative standard curve experiments e The best fit line in a plot of the Cy values from the standard reactions plotted against standard quantities See also regression line e A set of standards containing a range of known quantities Results from the standard curve reactions are used to generate the standard curve The standard curve is defined by the number of points in the dilution series the number of standard replicates the starting quantity and the serial factor Method for determining absolute target quantity in samples With the standard curve method the software measures amplification of the target in samples and ina standard dilution ser
229. identical sequence or they may differ somewhat often at one or more single base sites SNPs Display of genotyping data collected during the post PCR read The allelic discrimination plot is a graph of the normalized reporter signal from the allele 1 probe plotted against the normalized reporter signal from the allele 2 probe A segment of DNA amplified during PCR Part of the instrument run in which PCR amplifies the target Fluorescence data collected during amplification are displayed in an amplification plot and the data are used to calculate results Note Only quantitative real time PCR experiments not end point experiments take amplification data into account Calculation of the efficiency of the PCR amplification in an experiment EFF is calculated using the slope of the regression line in the standard curve A slope close to 3 32 indicates optimal 100 PCR amplification efficiency Display of data collected during the cycling stage of PCR amplification The amplification plot can be viewed as e Baseline corrected normalized reporter AR vs cycle e Normalized reporter Rp vs cycle e Threshold cycle Cr vs well Part of the instrument run in which PCR amplifies the target The amplification stage called a cycling stage in the thermal profile consists of denaturing primer annealing and extension steps that are repeated Fluorescence data collected during the extension stage are displayed in an amplification p
230. ies Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates the absolute quantity of target in the samples See also standard and standard curve In standard curve and relative standard curve experiments a set of standards containing a range of known quantities The standard dilution series is prepared by serially diluting standards For example the standard stock is used to prepare the first dilution point the first dilution point is used to prepare the second dilution point and so on In the software the volumes needed to prepare a standard dilution series are calculated by the number of dilution points the number of standard replicates the starting quantity the serial factor and the standard concentration in the stock See also standard curve In the PCR reaction a known quantity In standard curve experiments the quantity of target in the standard In the software the units for standard quantity can be for mass copy number viral load or other units for measuring the quantity of target Standard quantity can also refer to dilution factor When defining a standard curve in the software the highest quantity A component of the thermal profile For each step in the thermal profile you can set the ramp rate ramp increment for melt curve steps hold temperature and hold time duration You can turn data collection on or off for the ramp or the hold part
231. ifications and Layout 0c cee nn rnnr aranean nne 20 QuantStudio 12K Flex System specifications 000 cece eee e eee eens 20 QuantStudio 12K Flex System layout and connections n 0c eee e eee eee 23 QuantStudio 12K Flex System hardware 0 0 00 e cece cece eee e tenet eens 24 Instrument Components dX KX unce ot Pee Pee ee cee Mead cede 24 Barcode reader d iv goes pages Socks R R 9 cele ea ees cals Maney cate i ee nee Hee 26 Twister Robot component lt x Xe AST X aa ccc cence mien dK nee nee KAR es 27 Electrical protective devices cece eee eee nent enna 29 QuantStudio 12K Flex System Software n n 00200 cece eee eee e errr 30 Computer requirements 000cce cece eee cette eet ttetetet ee ee eeee 30 Software installation s s eee sarees ccc cece nde eee eden aa 30 Twister Robot software orses atacs denna K RA TRE AR Ra R e da TT 31 Third party Software 200 annarrar nrnna rnern enaena 31 QuantStudio 12K Flex System consumables 0 0000 cece cece eee tenet eeee 32 Compatible consumables 00 cece cece ete eee tet t eee eeeaee 32 Guidelines for handling consumables cece cee ee eeee 33 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 3 Contents S CHAPTER 2 Calibrating Multi Well Plate and Array Card Sample STs HT 35 Recommended calibration and maintenance 0 00 c eee eee eee ee ee 36 Preparing
232. ified fuses of the same type and rating as those currently in the instrument CAUTION PHYSICAL INJURY HAZARD Do not attempt to lift the instrument or any other heavy objects unless you have received related training Incorrect lifting can cause painful and sometimes permanent back injury Use proper lifting techniques when lifting or moving the instrument At least two people are required to lift the instrument CAUTION PHYSICAL INJURY HAZARD Do not remove the instrument cover There are no components inside the instrument that you can safely service yourself If you suspect a problem contact an Life Technologies Service Representative WARNING PHYSICAL INJURY HAZARD The QuantStudio 12K Flex System and LED are hot The LED can become very hot while in use Allow the LED to cool for 15 minutes and put on protective powder free gloves before handling it CAUTION PHYSICAL INJURY HAZARD During instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature CAUTION PHYSICAL INJURY HAZARD Wear disposable powder free gloves when handling the LED to prevent burns and to prevent shortening the life of the replacement LED Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 249 G Safety Safety alerts 250 Applied Biosystems QuantStudio 12K Flex R
233. il e QC Status Indicates the quality of the calibration data where passed indicates that all wells produced data that passed the quality check and failed indicates that one or more wells produced spectra that deviate significantly from the other wells on the plate Analysis ac Action status status Passed Passed Click Next then remove the plate or array card when the QuantStudio 12K Flex Instrument ejects the tray arm Passed Failed Troubleshoot the failed calibration as described in Troubleshooting background calibrations on page 73 Failed Failed Note You can accept a calibration that passes the Analysis Status check but fails the QC Status check We recommend using calibrations that yield passing results for both status reports WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the plate remove the plate according to Troubleshooting background calibrations on page 73 8 Discard or store the plate or array card Consumable Action Array card Discard the array card Plate Return the background plate to its packaging sleeve then return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate is stored in its original packaging sleeve you can use i
234. ile 228 sample file 206 forward primer 258 fuse cover 25 28 fuse replacement 114 G genotyping experiment 258 guidelines chemical safety 245 chemical waste disposal 246 chemical waste safety 247 consumable preparation 32 networking 128 OpenArray Calibration Cases handle 91 remote monitoring 132 TaqMan OpendArray plate handle 95 H hand held barcode reader 26 hard drive maintenance 110 hazard icons See safety symbols on instruments 238 hazard symbols See safety symbols on instruments hazards See safety heated cover 24 handling 121 installation 121 temperature setting 168 Help system accessing 252 heterozygote 258 holding stage 258 homozygote 258 housekeeping gene 258 humidity requirement 22 icon instrument 168 identifying contamination 80 107 iLink PRO Software 31 import 258 audit settings 153 file formats 200 security settings 153 user account settings 153 IMPORTANT description 14 installation category 242 firmware updates 116 heated cover 121 instrument fuses 114 license keys 117 network 126 operating system updates 115 plate adapter 119 123 software 30 software updates 116 specification 65 95 third party software 31 instrument 18 24 25 accessories 232 APIPA support 126 Autodiscovery 170 background calibration 45 84 control monitor over anetwork 126 data transfer 133 date time setting 168 DHCP support 126 dye calibration 53 90 184 electrical requirements
235. illed OpenArray Calibration Cases into the same positions on the OpenArray Calibration Carrier IMPORTANT The instrument should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 8 Repeat steps 1 7 to fill the remaining three OpenArray Calibration Cases IMPORTANT Confirm that the OpenArray Calibration Cases are in their original positions and that their plugs are oriented away from the A1 position as shown Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 93 Calibrating OpenArray Plate Sample Blocks Dye calibration 9 Click OK to close this dialog box then click START RUN in the Run screen to start the filled reading IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature Complete the IMPORTANT Wear powder free gloves while preparing the OpenArray calibration _ calibration cases 1 Verify the status of the calibration e Analysis Status Indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or the data it collected is unusable e QC
236. ilure of the computer or its hard drive s e Conserves space on the hard drive and optimizes performance Develop a data management strategy We recommend developing a strategy for managing the files produced by the QuantStudio 12K Flex Software Note Real time runs generate significantly more data than genotyping or presence absence experiments During 24 hrs of real time operation the QuantStudio 12K Flex System can generate more than 10 MB of data Check disk space TM If you perform real time experiments on your QuantStudio 12K Flex System check the amount of available space on your hard drive weekly When the hard drive is within 20 of maximum capacity transfer the older data to another storage device You can use the QuantStudio 12K Flex Instrument touchscreen to back up the instrument settings instrument name icon standby time out and cover idle temperature In the event that the QuantStudio 12K Flex Instrument settings are reset you can restore the settings from the backup See Backing up the QuantStudio 12K Flex Instrument settings on page 164 for more information 110 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 4 Maintenance 4 Decontaminating the sample block Decontaminating the sample block Perform this procedure to eliminate fluorescent contaminants from the QuantStudio 12K Flex System sample block Contaminat
237. in ul eg wo Z9 C9 ul gg WO 0 26 20 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Figure 2 OpenArray Accufill System 182 88 cm 72 in Chapter 1 Getting Started Specifications and layout 79 cm 31i 45 72 cm 18 in ul gL WZS Cu gz Wo y9 Cur o WO Z 91 E Cu 9 WO py L6 s Required clearances The QuantStudio 12K Flex System requires the following additional clearances Component Top Front Sides Back QuantStudio 12K Flex 30 48 cm 122 0 cm 51 0 cm 15 2 cm Instrument 12 0 in 48 0 in 20 0 in 6 0 in Twister Robot 15 2 cm 15 2 cm 15 2 cm 15 2 cm 6 0 in 6 0 in 6 0 in 6 0 in OpenArray Accufill System 190 0 cm 10 0 cm 76 0 in 4 0 in Computer and optional UPS 30 48 cm 15 24 cm 12 0 in 6 0 in Instrument hot air exhaust venting The maximum thermal output of the QuantStudio 12K Flex Instrument is 2731 BTU hr 800 W vented directly into the room air from the hot air waste port on the rear panel Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 21 22 Getting Started Specifications and layout Electrical requirements Note We recommend placing the QuantStudio 12K Flex Instrument
238. ing a QuantStudio 12K Flex Instrument 0 00008 132 IMPORTANT This chapter does not provide adequate detail to integrate the Applied Biosystems QuantStudio 12K Flex Real Time PCR System into all possible network architectures Because your network may contain advanced features such as a firewall or network domains we recommend that you consult a network administrator before connecting the QuantStudio 12K Flex System to your laboratory network Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 125 5 Networking Networking overview Networking overview Controlling and monitoring networked instruments About the Ethernet port TM After installing the QuantStudio 12K Flex System you can connect the QuantStudio 12K Flex System to a local area network to enhance its functionality This chapter describes how to e Set up the QuantStudio 12K Flex System for use on a network e Set up a computer for remote monitoring e Test the network connection by engaging the remote monitoring feature When the QuantStudio 12K Flex Instrument is connected to a network computers on the network that are running the QuantStudio 12K Flex Software can control or monitor it The QuantStudio 12K Flex Software can control up to 4 instruments and monitor up to 15 instruments simultaneously A networked QuantStudio 12K Flex Instrument can be controlled b
239. ing the network settings on page 170 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 129 5 Networking Connecting the computer to the network Connecting the computer to the network After connecting the QuantStudio 12K Flex Instrument to the network connect the computer to the network and install the QuantStudio 12K Flex Software for remote monitoring Required materials Ethernet cable with RJ45 connectors Computer If you are connecting a computer that you provided to a network confirm that the requirement computer contains a free network port Required Obtain the following information from your network administrator information e Network policy for obtaining IP addresses DHCP or static IP e Ifthe network requires static IP addresses obtain the IP address subnet mask and gateway address for the computer Setting up the IMPORTANT We recommend that you arrange for a network administrator to connect computer your computer to the network The following procedure does not provide adequate detail for all network architectures Note The following procedure is valid for the Microsoft Windows XP operating system 1 Use the Ethernet cable to connect the computer to the nearest network port 2 Power on the computer then log in using a user account that belongs to the Administrators user group 3 In the computer desktop right click My Network Places then se
240. io 12K Flex Software licenses on page 117 You can use the QuantStudio 12K Flex Instrument touchscreen to update the QuantStudio 12K Flex Instrument firmware See Updating the QuantStudio 12K Flex Instrument firmware on page 116 for more information 116 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 4 Maintenance 4 Managing QuantStudio 12K Flex Software licenses Managing QuantStudio 12K Flex Software licenses About QuantStudio 12K Flex Software license keys and files Managing licenses You can use the License Central feature to monitor activate or install the licenses that control access to the QuantStudio 12K Flex Software base application and associated modules The QuantStudio 12K Flex Software and associated modules require the installation and maintenance of valid license files for continued operation The license files are generated by the Life Technologies website when a license key is activated Each file pairs a software license key with the computer from which the key was activated After a key is activated and a license file is generated the file cannot be transferred to another computer To transfer a license between computers you must reactivate the license key using the QuantStudio 12K Flex Software on the target computer Note QuantStudio 12K Flex Software licenses are valid for a limited time and they
241. ion Plate Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Verifying the instrument performance The QuantStudio 12K Flex System passes the installation specification if the inequality holds and the QuantStudio 12K Flex Instrument successfully distinguishes between unknown populations A and B with a statistical confidence level of 99 7 As shown in the following table you can omit a limited number of outlier wells from the unknown populations to meet the installation specification Maximum number of outlier wells that can be removed Sample block Unknown Unknown Standards No template population Af population B STD controls NTC 96 well platett 6 6 1 0 384 well plate 10 10 2 0 Array card 4 4 4 0 T 5 000 copy population for 384 well plates 800 copy population for array cards 10 000 copy population for 384 well plates 1 600 copy population for array cards Maximum number of wells that can be removed from each standard population ttStandard 96 well plates or Fast 96 well plates IMPORTANT When performing the RNase P instrument verification experiment e Perform all calibrations beforehand s Run the TaqMan RNase P plate or array card soon after you allow the plate or reagents to thaw Minimizing the time between thaw and run ensures optimal performance e Wear powder free gloves and safety glasses when you prepare plates or array cards Prepare the instrument
242. ion factor dye calibration Dye Library EFF efficiency correction Algorithm used to determine the threshold cycle The software provides two Cy algorithms Baseline Threshold and Relative Threshold Dye that is not precalibrated for an instrument Custom dyes that fall within the emission wavelength range of the instrument can be added and adapted for use in experiments on the QuantStudio 12K Flex Instrument To use a custom dye add the dye to the Dye Library and perform a dye calibration See threshold cycle CT In the thermal profile a stage that is repeated A cycling stage is also called an amplification stage See also amplification stage See quantification cycle Cq During the instrument run a process in which an instrument detects fluorescence data from each well of the reaction plate The instrument transforms the signal to electronic data and saves the data in the experiment file In the QuantStudio 12K Flex Software a data collection point is indicated by an icon in the thermal profile e Data collection on e Data collection off Fl See baseline corrected normalized reporter DRn A reagent used to dilute a sample or standard before it is added to the PCR reaction See serial factor Type of calibration in which the software collects spectral data from a series of dye standards and stores the spectral information for the dye standards in a pure spectra calibration file This file is used during
243. ion is generally evident in failed background calibrations where one or more wells consistently exhibit abnormally high signals CAUTION PHYSICAL INJURY HAZARD Do not remove the QuantStudio 12K Flex Instrument cover There are no components inside the instrument that you can safely service yourself If you suspect a problem contact a Life Technologies Service Representative CAUTION PHYSICAL INJURY HAZARD During instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature CAUTION Before using a cleaning or decontamination method other than those recommended verify with Life Technologies that the proposed method will not damage the equipment Required materials s Bleach 10 solution e Tissue lint free e Cotton or nylon swabs and lint free cloths e Ethanol 95 solution e Safety glasses e Pipette 100 uL with pipette tips e Powder free gloves e Screwdriver s Deionized water Handling the To prevent damaging or contaminating the sample block sample block handle the assembly as shown Also when the assembly has been removed from the QuantStudio 12K Flex Instrument place the sample block on a clean dry surface or in its shipping container Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 111 7 Maintenance Decontaminating the sample block
244. ions WARNING CHEMICAL HAZARD All chemicals in the instrument are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument T WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles i WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety guidelines To minimize the hazards of chemicals Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For a
245. ions from the touchscreen to operate the QuantStudio 12K Flex Instrument without using the QuantStudio 12K Flex Software m Creating an experiment from a template e 0 eee eee 159 E Running an experiment ag KR 0 K 00 E KR eee eee tenes 160 m Transferring experiments templates and results data 6 161 Note If the QuantStudio 12K Flex Instrument is operating in secure mode see Enabling disabling instrument security on page 172 users can only open and close the side door Creating an 1 If necessary download the experiment template to the QuantStudio 12K Flex experiment from a Instrument as described in Transferring experiments from a USB drive on template page 161 2 Ifthe instrument is in standby touch the QuantStudio 12K Flex Instrument touchscreen to activate it then press G 3 In the Main Menu touch G View Templates 4 Inthe View Templates screen touch a template then touch New To view the parameters of a template select the desired template then touch View When finished touch to return to the View Templates screen Note You cannot modify the experiment parameters of a template 5 In the Create New Experiment screen touch each field to set the e Touch the New Experiment Name field and use the keypad to enter a name up to 100 characters for the experiment e Touch the Save to Folder field to open the Select Folder Screen e Touch the Reaction Volume
246. ired Specifies the format of the exported data see RDML export format on page 228 for the export file specifications QuantStudio12KFlex Exports data in a format compatible with the QuantStudio 12K Flex System SDS23 Exports data in a format compatible with the Applied Biosystems 7900HT Real Time PCR System RDML Exports data in the real time data markup language RDML format Example f RDML l lt path gt Optional Specifies the path lt path gt of the directory to which the software saves the exported files Example 1 C exports s lt option gt Optional Specifies the data spanning option lt option gt that determines how the software exports data from multiple experiments single Exports data for all experiments into one contiguous data file multiple Exports data for each experiment to a separate data file Example s multiple x lt filepath gt Required Specifies the file format of the exported file QuantStudio12KFlex export format txt xls or xlsx SDS23 export format txt RDML export format rdml Example x rdml 196 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix D Command line Software Operation D Examples Examples Batch file creation The following example uses all parameters described in Command syntax and arguments on page 194 required and option
247. is installed only with Applied Biosystems QuantStudio 12K Flex Real Time PCR Systems that were purchased with the SAE module IMPORTANT Enabling the Security Audit and Electronic Signature module alone does not make the QuantStudio 12K Flex System compliant with any particular standard You must modify the module settings according to your requirements to ensure compliance The Security Audit and Electronic Signature SAE module is an optional component of the QuantStudio 12K Flex Software that can allow you to configure the QuantStudio 12K Flex System to meet specific requirements The module provides the following functionality e Security Controls user access to the software A default Administrator user account is provided and additional user accounts and permissions can be user defined Note The default password for the Administrator user account is Administrator however the password can be changed during installation Note You can enable or disable system security globally e Auditing Tracks changes made to library items actions performed by users and changes to the Security Audit and Electronic Signature settings The software automatically audits some actions silently You can select other items for auditing and specify the audit mode The Auditing function provides reports for audited library items Security Audit and Electronic Signature changes and actions Note You can enable or disable
248. it back into the plate retainer black side up IMPORTANT Do not flip the OpenArray Calibration Plaque over The black side of the tool must face up Click OK to close this dialog box then click START RUN in the Run screen to perform the second reading Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 85 Calibrating OpenArray Plate Sample Blocks Background calibration Complete the IMPORTANT Wear powder free gloves and safety glasses when you handle the calibration OpenArray Calibration Plaque 1 Verify the status of the calibration The Analysis Status displayed by the QuantStudio 12K Flex Software indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or the data it collected is unusable Analysis status Action Click Next 2 Enter any comments you have in the Comments field click Finish then click Yes when prompted to save the results Passed Failed 1 Repeat the calibration If necessary clean the OpenArray Calibration Plaque before you repeat the calibration as described in Caring for the OpenArray Calibration Plaque on page 83 2 If the calibration fails again contact Life Technologies for further assistance When the instrument door opens remove the OpenArray Calibration Plaque f
249. ive Reference The following Plate Setup values take precedence over the template e Block Type e Well Quantity to Well Assignment e Target Assay Marker to Well Assignment e Sample Color e Sample to Well Assignment e Biological Group Color e Task to Well Assignment e Target Color e Biological Group to Well Assignment 192 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix D Command line Software Operation DI Running the command line application Running the command line application Running the 1 In the desktop select Start Run application 2 In the Run dialog box enter cmd in the Open field then click OK 3 In the DOS prompt change to the installation directory and enter the command a Enter cd C Program Files Applied Biosystems QuantStudio12KFlex bin then press Enter b Enter cmdlineutil exe followed by expgen or export then all applicable parameters and arguments See Command syntax and arguments on page 194 for a complete list of command line parameters Viewing the The command line application includes a help function that provides the information command line help in this chapter To view help for e The entire application enter cmdlineutil exe help e A particular function enter cmdlineutil exe expgen help to view the file generation help or cmdlineutil exe export help to view the file export help Applied Biosystems Quant
250. king 5 Monitoring a QuantStudio 12K Flex Instrument Modifying the notification settings for a monitored instrument 1 Open the Calibration Reminders screen for the QuantStudio 12K Flex Instrument a In the Home screen of the QuantStudio 12K Flex Software click Instrument Console b In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it c After the QuantStudio 12K Flex Instrument is added to your list select it then click Manage Instrument d In the Instrument Manager click Maintenance then click Calibration Reminders 2 In the Calibration Reminders Setting table configure the notification settings for the calibrations in interest For each calibration that you want to monitor a In the Expiry Interval column enter the number of days that elapse before the type of calibration expires on the QuantStudio 12K Flex Instrument b In the Send a Reminder column select the check box to configure the QuantStudio 12K Flex Software to email a reminder to perform the calibration c Inthe Reminder Interval column enter the number of days that elapse before the software emails recipients a reminder to perform the calibration 3 In the Enter e mail addresses for notifications field enter the email address es tha
251. l Float Ct Mean The average C of the replicate wells for the specified target sample combination Float Ct SD The standard deviation of the average Cy of the replicate wells for the specified target Float Quantity s Unknown wells The calculated quantity for the sample at the well e Standard wells The quantity assigned to the standard at the well Float Quantity Mean e Unknown wells The average quantity of the replicate wells for the target sample e Standard wells The quantity assigned to the replicate wells for the target sample Float Quantity SD The standard deviation of the average quantity of the replicate wells for the target sample Float Automatic Ct Threshold Whether the threshold was determined automatically true or manually false true Or false Ct Threshold The threshold cycle C7 for the sample at the well Float Automatic Ct Whether the baseline was determined true Or false Baseline automatically true or manually false Baseline Start The first cycle used to calculate the baseline Integer Baseline End The last cycle used to calculate the baseline Integer Tm1 Tm3 The first second and third melting temperatures Float Tal calculated in degrees Celsius Comments Additional text that describes the well 1024 character string Custom The contents of the custom text fields found inthe 1024 char
252. l reagents are contained in the bottom of the wells The calibration plates must be well mixed and centrifuged before use Plates only Do not discard the packaging for the calibration plates Each plate can be used to calibrate the QuantStudio 12K Flex System 3 times for up to 6 months if it is stored in its packing sleeve Plates only Handle the calibration plates with care to prevent contamination Do not place the plates on a lab bench to avoid contaminating them Always put calibration plates back into their packaging sleeves 96 well plates only If you are using cap strips to seal your plates firmly seal all wells before running the plate Partially seated caps can leak during the experiment causing evaporation Tubes only Firmly seal all individual tubes and tube strips Partially seated caps can leak during the experiment causing evaporation OpenArray plates only Hold OpenArray plates by the edges of the cases Do not touch the through holes OpenArray plates only Load and seal the TaqMan OpenArray plate within one hour after opening the plate packaging OpenArray plates only If you drop a loaded OpenArray plate discard it in the appropriate waste container Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 33 1 Getting Started QuantStudio 12K Flex System consumables 34 Applied Biosystems QuantStudio 12K Flex Real Time PCR System
253. l the software to the computer that contains the QuantStudio 12K Flex Software 2 Perform several dry run test experiments using plates that do not contain reagents Note The goal of the test experiments is to run plates under conditions that match normal instrument operation Therefore the characteristics of the test experiments plate layout and run method must closely resemble your actual experiments 3 Confirm that the QuantStudio 12K Flex System performs each test experiment without producing errors If the QuantStudio 12K Flex System performs the tests successfully proceed with your experiments If the QuantStudio 12K Flex System encounters errors during the test runs the software may not be compatible with the QuantStudio 12K Flex Software Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 31 1 Getting Started QuantStudio 12K Flex System consumables QuantStudio 12K Flex System consumables Compatible The QuantStudio 12K Flex System supports a series of specialized consumables consumables through interchangeable sample blocks Use the consumables appropriate for the sample block of your QuantStudio 12K Flex System Sample Pan zabi Reaction block volume 96 well plate e MicroAmp Optical 8 Cap 50 uL 0 2 mL Strip e MicroAmp 8 Tube Strips 0 2 mL e MicroAmp Reaction Tubes without Caps 0 2 mL s
254. lates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained Note The OpenArray plate can be loaded into any position on the plate carrier 6 After loading the OpenArray plate start the calibration a In the Overview screen select Check the box when the RNase P calibration plate has been loaded then click Next b In the Run screen click START RUN to start the calibration IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature 7 When the run is complete and the QuantStudio 12K Flex Software displays the Analysis screen verify the status of the run Analysis status Action Passed Go to step 12 on page 104 Failed Go to step 8 to review the data for outliers If the run fails the QuantStudio 12K Flex Software may have included outliers that caused the initial analysis to fail Experimental error may cause some through holes to be amplified insufficiently or not at all These through holes typically produce Cy values that differ significantly from the average for the associated replicate through holes If included in the calculations these outlying data outliers can result in erroneous measurements
255. le block maintenance Frequency Maintenance task Weekly Check the computer disk space If necessary archive or back up your experiment files and instrument settings Power off the computer that controls the QuantStudio 12K Flex System then after 30 seconds power on the computer Clean the surface of the QuantStudio 12K Flex System with a lint free cloth Perform a QuantStudio 12K Flex Instrument self test Monthly Perform a background calibration T Run disk cleanup and disk defragmentation Annually Perform a background calibration Perform a uniformity calibration Perform a dye calibration Perform an instrument verification run As needed Decontaminate the QuantStudio 12K Flex System Replace the QuantStudio 12K Flex System fuses Update the Windows operating system Update the QuantStudio 12K Flex Software and firmware T You can perform a background calibration to check for contamination If any parts of the optics are replaced or moved you must perform all calibrations including an RNase P instrument verification run 82 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 3 Calibrating OpenArray Plate Sample Blocks 3 About the OpenArray Calibration Plaque About the OpenArray Calibration Plaque The OpenArray Calibration Plaque is a specialized tool t
256. le locks into place IMPORTANT Confirm that the indicator on the left side of the sample block is positioned behind the red line on the instrument rail If the indicator is forward of the red line push the sample block into the instrument until it is seated correctly If the ROI calibration continues to fail check the status of the LEDs within the QuantStudio 12K Flex System then replace the LEDs if necessary Instrument malfunction Multiple possible causes Contact a local Life Technologies Field Service Office Instrument does not eject the ROI plate The adhesive cover may have adhered the plate to the heated cover within the instrument 1 2 3 Power off the QuantStudio 12K Flex Instrument Wait for 15 minutes then power on the instrument and eject the plate If the plate does not eject power off and unplug the QuantStudio 12K Flex Instrument then open the access door Wearing powder free gloves reach into the QuantStudio 12K Flex Instrument and remove the plate from the heated cover then close the access door Perform a background calibration to confirm that the sample block has not been contaminated k Bunooysayqnos sy20 g ajdwies pueg esy pue ajejq N M MNW Buyewqnes 0IDHIG1UREnpH swas soig panddy wl aping Jas uoOlessiulwpy pue adueuajulep WajSkS Yd aw jeay X YZ EL Table 2 Troubleshooting background calibrations Problem symptom
257. lect Properties 4 Right click Local Area Connection then select Properties 5 Select Internet Protocol TCP IP then click Properties 130 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 5 Networking 5 Connecting the computer to the network 6 Set the Internet Protocol TCP IP Properties for either DHCP or Static IP communication Network R 3 Action configuration DHCP Select Obtain an IP address automatically 2 Set the DNS address If the computer obtains DNS addresses e Automatically Select Obtain DNS server address automatically e Statically Select Use the following DNS address enter the address of the preferred and alternate DNS servers if available Static IP 1 Select Use the following IP address 2 In the IP Address field enter the static IP address 3 If necessary enter a subnet mask 4 If necessary enter a static gateway address in the Default Gateway field 7 If your network requires advanced TCP IP setup such as WINS define the settings a Click Advanced in the Internet Protocol TCP IP Properties dialog box b Define the IP Settings DNS and WINS tabs as instructed by your systems administrator then click OK 8 Close all dialog boxes by clicking OK 9 Restart the computer The computer is now visible to other computers on the network Installing the 1 If you are using a compu
258. libration 83 274 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide plate background calibration 46 dye calibration 55 layout 261 normalization calibration 59 preparation guidelines 32 RNase P instrument verification 65 ROI calibration 42 50 signing 155 plate adapter 24 installation 123 Plate Holder 97 plate setup file 261 file format 201 plates calibration 32 pollution requirement 22 port Ethernet 25 126 129 RS232 serial 25 28 USB 25 positions robot racks 28 positive control 261 post PCR read 261 power LED 27 port 25 28 requirements 22 switch instrument 25 power line regulator 29 power on off the instrument 176 177 prepare array cards 37 66 background calibration plate 46 custom dye plate 185 dye calibration plates 55 normalization calibration plate 59 plate for instrument verification 65 96 RNase P experiment 65 96 ROI calibration plate 42 50 pre PCR read 261 primer mix 261 primer probe mix 261 print action logs 146 149 152 audit reports 148 user report 145 protective devices electrical 29 Index pure dye 261 purge audit records 148 Q quantification method 262 quantity 262 QuantStudio export file format 209 quencher 262 QuickStart 262 R R2 value 68 racks robot 28 radioactive waste handling 247 ramp 262 ramp speed 262 raw data plot 262 RDML 262 RDML export file format 228 reaction mix 262 reagents 262 real time PCR
259. licenses software 117 Life Technologies support 252 line conditioner requirements 29 loading OpenArray plate 99 location requirement 22 log in user account 154 log instrument 173 logged in user name display 143 in user account 142 M made to order assays 259 maintenance background calibration 45 84 computer hard drives 110 dye calibration 53 90 experiment files 110 instrument 163 instrument settings 110 164 normalization calibration 59 reminders 169 RNase P instrument verification experiment 63 95 ROI calibration 41 schedule 36 82 software licenses 117 uniformity calibration 49 87 manual baseline 259 manual CT 259 materials accessories 232 consumables 236 kits 233 mDNS support 126 melt curve 259 melting temperature Tm 259 Microsoft NET Framework 31 Microsoft Data Access Components MDAC 31 Microsoft SQL 2005 Manager 31 Microsoft VBA Service Packs 31 monitoring instrument over anetwork 126 monthly maintenance tasks 36 82 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 273 Index moving and lifting safety computers and monitors 241 instrument 241 moving parts safety 242 moving the instrument 179 multicomponent plot 259 N name instrument 168 NED dye 19 53 negative control NC 260 negative control blocked IPC wells 260 negative control IPC wells 260 network computer setup 130 guidelines 128 instrument setup 129 layouts
260. ll for the target or SNP assay Available tasks e Unknown e Negative Control e Standard standard curve and relative standard curve experiments e Positive control genotyping experiments e IPC presence absence experiments e Blocked IPC presence absence experiments Wells containing identical reaction components including sample important for evaluating precision In the software a display of temperatures for the instrument cover and instrument block during the instrument run The type of nucleic acid to add to the PCR reaction A user created file that contains experiment setup information experiment type sample names target name and thermal conditions to be used as a starting point for new experiment setup Template files have an edt extension Part of the run method that specifies the temperature time ramp and data collection points for all steps and stages of the instrument run Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 267 Glossary threshold threshold cycle Cy Tm touchscreen uniformity calibration unknown unknown IPC wells y intercept e In amplification plots the level of fluorescence above the baseline and within the exponential growth region For the Baseline Threshold algorithm the threshold can be determined automatically see automatic threshold or can be set manually see manual threshold e In presence absen
261. lot and the data are used to calculate results With TaqMan chemistry the last two steps of a PCR stage are typically combined See also cycling stage In the software a collection of analysis settings to use in experiments You can save settings and reuse them You cannot edit or import settings into the library Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 253 Glossary assay Assay ID assay information file AIF assay mix AutoDelta automatic baseline automatic threshold background calibration baseline In a PCR reaction mix two target specific primers or two primers and a probe used to amplify a target Identifier assigned by Life Technologies to TaqMan assays Tab delimited data file on a CD shipped with each assay order The AIF contains technical details about all assays in the shipment It includes information about assay concentrations reporters and quenchers used part and lot numbers and assay vial and plate ID numbers The file name includes the number from the bar code on the plate PCR reaction component in Applied Biosystems TaqMan assays The assay mix contains primers designed to amplify a target and a TaqMan probe designed to detect amplification of the target In the run method a setting to increase or decrease the temperature and or time for a step with each subsequent cycle in a cycling stage When AutoDelta is enabled
262. lowing issues e Leading and trailing spaces in user names are difficult to detect on the screen or in printed reports e The number of consecutive spaces in a user name is difficult to determine on the screen or in printed reports Spaces in user names may cause confusion when a user searches for an audit record associated with a user name To find a record associated with a user name specify the user name exactly including leading consecutive and trailing spaces In the QuantStudio 12K Flex Software select Tools gt Security gt Settings In the Security screen click Set Up Messaging Notifications to display the Setup Notifications dialog box Select the events for notification e System security enabled or disabled Security has been enabled or disabled e User did not enter correct password A user attempts to log in with an incorrect password The message indicates the number of failed authentications e User account suspended The user exceeds maximum number of allowed failed authentications login attempts with an incorrect password e User session timed out No activity occurred in a user account for the specified period of inactivity 140 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 6 Security Audit and Electronic Signature 6 Configuring the system security 4 Select the notification method e Notify Admin at Login
263. m or select Start All Programs gt Applied Biosystems gt QuantStudio 12K Flex System gt QuantStudio 12K Flex Software d If the QuantStudio 12K Flex Software Login appears enter your user name and password then click OK 176 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix B Powering On or Off Storing and Moving the System Powering off the QuantStudio 12K Flex System Powering off the QuantStudio 12K Flex System The Applied Biosystems QuantStudio 12K Flex Real Time PCR System operates in low power mode when not in use however the QuantStudio 12K Flex System can be powered off completely so that the components draw no power Note If the QuantStudio 12K Flex System will be inactive for extended period of time prepare it for storage as explained in Storing the QuantStudio 12K Flex System on page 178 To power off the QuantStudio 12K Flex System components 1 TM Power off the QuantStudio 12K Flex Instrument a If the QuantStudio 12K Flex Instrument touchscreen is not blank touch to place the QuantStudio 12K Flex Instrument into stand by mode b Toggle the power button on the rear of the QuantStudio 12K Flex Instrument 2 Power off the QuantStudio 12K Flex System computer a In the desktop select Start Shut Down b In the Shut Down Windows dialog box select Shut Down then click OK 3 Power
264. mple block and plate adapter to match the new consumable format Close the access door Confirm that the access door is completely closed The QuantStudio 12K Flex Software displays an error message if the door is not completely closed and latched or if the sample block is not seated correctly Plug in and power on the QuantStudio 12K Flex System In the Home screen of the QuantStudio 12K Flex Software click Instrument Console In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments then review the Heated Cover Firmware Version field in the Instrument Properties pane The installation is successful if the QuantStudio 12K Flex Instrument powers on and if the Heated Cover Firmware Version field displays a version number Perform the following calibrations in the specified order ROI calibration Background calibration Uniformity calibration Dye calibration then Normalization calibration Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 4 Maintenance Replacing the plate adapter Replacing the plate adapter Replace the plate adapter in the event of a hardware failure or if you want to change the consumable format of the QuantStudio 12K Flex Instrument WARNING PHYSICAL INJURY HAZARD During instrument operation the sample block and heated cover can be heated to 100 C Before removing the he
265. n calibration plate or page 50 plates or go to Performing the calibration on page 51 array cards array card Otherwise Prepare the ROI calibration consumable appropriate for your QuantStudio 12K Flex Instrument m Preparing the ROI calibration plate 0 cece eee 42 E Preparing array cards for instrument calibration lt rnn 37 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 49 Calibrating Multi Well Plate and Array Card Sample Blocks Uniformity calibration Preparing the IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or calibration plate array cards Required materials See ROI calibration on page 41 for a complete list of materials for the calibration Preparing the ROI calibration plate 1 Remove the ROI calibration plate from the freezer then allow it to warm to room temperature approximately 5 minutes IMPORTANT Do not remove a calibration plate from its packaging until you are ready to run it The fluorescent dyes in the wells of the plate are photosensitive Prolonged exposure to light can diminish the fluorescence of the dye 2 Remove the ROI calibration plate from its packaging Do not remove the optical film IMPORTANT Do not discard the packaging for the calibration plate You can use the plate to calibrate a QuantStudio 12K Flex System 3 times for up to 6 months if it is stored in its slee
266. n 37 array cards for verification 63 custom background plate or array card 182 custom dye plate 185 experiments from the instrument 159 Ct algorithm 256 custom dyes 19 54 256 add to software 186 calibration 184 create plate 185 cycle threshold 255 256 cycling stage 256 D DANGER description 14 data background calibration 45 84 dye calibration 54 91 normalization calibration 59 ROI calibration 41 transfer to from the instrument 133 161 uniformity calibration 49 87 data collection 18 256 data management 110 date time instrument 168 decontamination identify contaminants 80 107 sample block 111 delta Rn 256 DHCP support 126 diluent 256 dimensions instrument 20 disable calibration reminders 134 security instrument 172 security software 139 270 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide DNS support 126 documentation related 251 door access 24 side 24 dye calibration 53 90 256 data 54 91 perform 56 91 spectra evaluation 55 troubleshoot 75 when to perform 53 90 Dye Library 256 dyes custom 19 54 system 19 53 91 E efficiency correction 256 electrical protective devices 29 electrical requirements 22 electrical safety 242 electromagnetic compatibility standards See EMC standards electronic signature administrators actions that allow e sig 151 enable or disable 150 functions that require e sig 151 is signed field 155
267. n Dye Kit TaqMan Array Calibration with FAM Dye TaqMan Array Calibration with VIC Dye TaqMan Array Calibration with ROX Dye TaqMan Array Calibration with RO Dye TaqMan Array Calibration with FAM ROX Dye TaqMan Array Calibration with VIC ROX Dye TaqMan Array Background Buffer 4432376 Kit TaqMan RNase P Array Card Instrument Verification Reagents Port 1 NTC Port 2 Unknown A Port 3 Unknown B Port 4 Standard 200 Copies Port 5 Standard 400 Copies Port 6 Standard 800 Copies Port 7 Standard 1600 Copies Port 8 Standard 3200 Copies 44322654 15 to 25 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 235 Parts and Materials Consumables Consumables Note For consumable shelf life expiration date see the package label The following consumables are to be used with the Applied Biosystems QuantStudio 12K Flex Real Time PCR System QuantStudio 12K Flex System consumable Part no Applied Biosystems Array Card Staker Sealer 4331770 Array Card Bucket Clip Set 1st Generation 4337762 2nd Generation 4442571 Array Cards 8 Port Empty Empty Array Card Kit 4 pk 4334812 Empty Array Card Kit 4351471 Centrifuge Buckets Array Card 1st Generation 4337230 2nd Generation 4442573 Clip Array Card Centrifuge Adapter 4334682 MicroAmp Fast 8 Tube Strip
268. n at least once per year or more often depending on use IMPORTANT You must perform a uniformity calibration before a dye calibration During the uniformity calibration the QuantStudio 12K Flex Software captures a series of images of the orange side of the OpenArray Calibration Plaque using each instrument filter using each instrument filter The QuantStudio 12K Flex Software uses the captured images to calibrate the optical uniformity of the QuantStudio 12K Flex Instrument IMPORTANT A user must be present throughout the duration of the calibration Following the first read the OpenArray Calibration Plaque must be rotated 180 degrees before the instrument can complete the calibration Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 87 Calibrating OpenArray Plate Sample Blocks Uniformity calibration Load the plaque 1 When the instrument door opens load the OpenArray Calibration Plaque orange side up into the plate retainer IMPORTANT Ensure that the OpenArray Calibration Plaque is loaded into the plate retainer so that the orange side of the tool is facing up IMPORTANT The instrument should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained Start the calibration a Select Check the box when the calibration plaque has been loaded then click Next b In t
269. n the rear of the QuantStudio 12K Flex Instrument Power off the computer a Select Start gt Shut Down b In the Shut Down Windows dialog box select Shut Down then click OK Power off the monitor If you have an Applied Biosystems Twister Robot toggle the power button on the rear of the Twister Robot 178 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix B Powering On or Off Storing and Moving the System Moving the QuantStudio 12K Flex System Moving the QuantStudio 12K Flex System Required materials Handling the sample block and heated cover Preparing the QuantStudio 12K Flex System components Perform this procedure to safely move the QuantStudio 12K Flex System short distances for example between laboratories of the same building CAUTION PHYSICAL INJURY HAZARD Do not attempt to lift the QuantStudio 12K Flex Instrument or any other heavy objects unless you have received related training Incorrect lifting can cause painful and sometimes permanent back injury Use proper lifting techniques when lifting or moving the QuantStudio 12K Flex Instrument At least two people are required to lift it IMPORTANT Moving your QuantStudio 12K Flex System can create subtle changes in the alignment of the instrument optics Recalibrate the instrument if necessary None To prevent damaging or contaminating the sample block or th
270. n the system you are prompted to overwrite or skip each account Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 153 Security Audit and Electronic Signature Users overview Users overview Security Logging in Permissions Changing your password when it expires Account suspension The Security Audit and Electronic Signature SAE module is an optional component of the QuantStudio 12K Flex Software The module provides the following functionality e System security Controls user access to the software e Auditing Tracks changes made to library items actions performed by users and changes to the Security Audit and Electronic Signature settings e Electronic signature Requires users to provide a user name and password when performing certain functions Depending on the way that your administrator configures these features you may see the following dialog boxes and prompts when you use the software If security is enabled on your system you must provide a user name and password to access the software Your access to functions in the software is based on the permissions associated with your user account Functions for which you do not have permissions are grayed Note If the QuantStudio 12K Flex Software is configured for password expiration you are periodically prompted to change your password Note If the QuantStudio 12K Flex S
271. n well e OFFSCALE Fluorescence is offscale s OUTLIERRG Outlier in replicate group e SPIKE Noise spikes e THOLDFAIL Thresholding algorithm failed 10 If you omitted outliers click Reanalyze to analyze the run If the status of the RNase P Run is Failed after performing steps 8 through 10 repeat the RNase P experiment using a different RNase P plate If the problem persists contact Life Technologies 11 Review the standard curve a Select the Standard Curve tab b Click the upper left corner of the Plate Layout to select all wells c Verify that the R2 value is 2 0 990 If the R2 value is less than 0 990 repeat the RNase P experiment using a different RNase P plate If the problem persists contact Life Technologies 12 In the Analysis screen click Next remove the plate or array card when the QuantStudio 12K Flex Instrument ejects the tray arm then discard the plate or array card WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the plate remove the plate according to Table 7 Troubleshooting RNase P instrument verification experiments on page 77 13 Click Finish then click Yes when prompted to save the experiment 70 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide
272. natures required for access When authorized persons use this feature they are creating a legally binding signature In the software a numeric value that defines the sequence of quantities in the standard curve The serial factor and the starting quantity are used to calculate the standard quantity for each point in the standard curve For example if the standard curve is defined with a serial factor of 1 10 or 10X the difference between any two adjacent points in the curve is 10 fold Regression coefficient calculated from the regression line in the standard curve The slope indicates the PCR amplification efficiency for the assay A slope of 3 32 indicates 100 amplification efficiency See also amplification efficiency EFF and regression line Single nucleotide polymorphism The SNP can consist of a base difference or an insertion or deletion of one base Used in genotyping experiments a PCR reaction that contains primers to amplify the SNP and two probes to detect different alleles In the software an editable collection of SNP assays to add to genotyping experiments The SNP assays in the library contain the SNP assay name SNP assay color and for each allele the allele name or base s reporter quencher and allele colors The SNP assays in the library may also contain the assay ID and comments about the SNP assay In the thermal profile a group of one or more steps Examples PCR stage cycling stage also called amplif
273. nce functions are for users who maintain the instrument and administration functions are for systems administrators or for information technology personnel The right most column indicates whether a function is available when the QuantStudio 12K Flex Instrument is operating in secure mode see Enabling disabling instrument security on page 172 for more information S Available in User role Function secure mode Operational Create experiments from templates No Run experiments Transfer experiments templates and results to from a USB drive Maintenance Back up and restore the instrument settings Yes Perform an instrument self test Update the QuantStudio 12K Flex Instrument firmware Administration Define the date and time Define the instrument settings Define the network settings Define the maintenance reminders Define the system shortcuts Enable or disable instrument security Review the instrument statistics View the QuantStudio 12K Flex Instrument log 158 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix A Manual Instrument Operation Operating the instrument from the touchscreen Operating the instrument from the touchscreen The touchscreen provides limited control of the QuantStudio 12K Flex Instrument to run experiments and transfer data You can perform the following funct
274. nd Tip e OpenArray Plate Press e Pipettes e Powder free gloves e Safety glasses Perform a dye calibration at least once per year or more often depending on use IMPORTANT You must perform a background calibration before every dye calibration Because the age and use of instrument components can affect spectra readings we recommend performing a dye calibration at least every year 90 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide About the dye calibration Guidelines for handling the OpenArray Calibration Cases Perform the empty reading Chapter 3 Calibrating OpenArray Plate Sample Blocks Dye calibration The dye calibration is a two part procedure in which the QuantStudio 12K Flex Instrument performs two readings of the OpenArray Calibration Cases e A preread of the empty OpenArray Calibration Cases e A postread of the OpenArray Calibration Cases filled with OpenArray FAM Dye Solution About the dye calibration data The product of the dye calibration is a spectral profile that represents the fluorescence signature of the FAM dye standard The profile consists of a set of spectra that correspond to the fluorescence collected from the OpenArray calibration cases The QuantStudio 12K Flex Software plots the resulting data for the spectral profile in a graph of fluorescence versus filter When the QuantStudio 12K Flex Software
275. nd VIC dye The following figure shows the emission spectrum for each dye and the filters and wavelengths at which each dye is read 7 S S oe O l l Wavelength nm 5 E Emission Spectra Dyes 520 nm 550 nm 580 nm 610 nm FAM VIC NED ROX SYBR Green TAMRA Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 53 54 Calibrating Multi Well Plate and Array Card Sample Blocks Dye calibration Custom dyes The QuantStudio 12K Flex System can be used to run assays designed with custom dyes not supplied by Life Technologies however before using custom dyes with the QuantStudio 12K Flex System you must create and run a custom calibration plate The QuantStudio 12K Flex Software uses the custom calibration plate to create a spectral standard to distinguish the custom dye in the fluorescence data collected during the run See Creating a custom dye plate for calibration on page 184 for information on custom dye calibrations IMPORTANT A custom dye must excite between 455 and 672 nm and read between 505 and 723 nm About the dye calibration data The product of a dye calibration is a collection of spectral profiles that represent the fluorescence signature of each dye standard Each profile consists of a set of spectra that correspond to the fluorescence collected from the wells of the spectral calibration plate The QuantStudio 12K Flex Softwa
276. nd temperature plots in real time view the calibration status perform calibrations and manage files on the instrument including downloading completed experiments to your computer Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide internal positive control IPC inventoried assays IPC IPC blocking agent IPC made to order assays manual baseline manual threshold melt curve melt curve characteristics melt curve stage melting temperature Tm melting transition region multicomponent plot Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Glossary In presence absence experiments a short synthetic DNA template that is added to PCR reactions The IPC can be used to distinguish between true negative results the target is absent in the samples and negative results caused by PCR inhibitors incorrect assay setup or reagent or instrument failure TaqMan Gene Expression Assays and TaqMan SNP Genotyping Assays that have been previously manufactured passed quality control specifications and stored in inventory See internal positive control IPC Reagent added to PCR reactions to block amplification of the internal positive control IPC See negative control IPC wells TaqMan Gene Expression Assays that are manufactured at the time of order Only assays that pass manufacturing quali
277. ng 4 Click Apply Settings Deleting a meaning 1 Select the meaning from the e Signature Meanings list then click Remove 2 Click Apply Settings To determine the user roles that can perform an electronic signature 1 In the e Signature tab of the Security Settings dialog box select the check box next to the appropriate user roles in the User Role signature rights table 2 Click Apply Settings IMPORTANT Do not change electronic signature settings during calibration 1 In the Signature Required column select the check box next to each action for which you want to require electronic signatures see below This selection causes the software to present an e sig prompt if a user performs the action on a data file that does not have the required signatures The data must be signed before the user can perform the action The QuantStudio 12K Flex Software requires Action Sa electronic signatures when a user Print Report Prints a report from an experiment Initiates a run from the QuantStudio 12K Flex Software or QuantStudio 12K Flex Instrument Start Run 2 For each meaning of each selected action enter the number of e signatures from each user role that are required before the software can execute the associated action For example in the following figure at least two users from the Administrator user role must sign an experiment using the Reviewed and Approved Plate Set Up meani
278. ng before a user can start the associated run Actions requiring signatures Signature Required Action ee El Meaning Administrator Technician Scientist Start Run S Hilin Reviewed and Approved Plate Set Up 2 o 0 Reviewed and Approved Results 0 0 0 Reviewed and Approved Template 0 0 0 3 Click Apply Settings Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 151 6 Security Audit and Electronic Signature Generating electronic signature reports How the software prompts electronic signature If the system is configured to check for a signature before starting a run or printing a report and the data are not signed the QuantStudio 12K Flex Software displays a message when the user clicks Start Run or Print Report Example The e signature system is configured to require signatures from two users from the user account named Administrator before a user can start a run The experiment has not been signed A user attempts to begin the run The following message is displayed Missing Required Signatures i The requested operation cannot be performed because some required signatures are missing The following e Signatures must be collected before this operation can be performed Reviewed and Approved Plate Set Up by 2 Administrator s Before the run can start two administrators must sign If a user with an incorrect user role
279. ng contamination rrara aa ae a cece teen eens 107 Viewing the ROX image files 0 0000 cece cece eee rreraren 108 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 5 Contents M CHAPTER 4 Maintenance 00 cece eee eee eee 109 Regular data maintenance nrn cece eee eee eee e enna 110 Maintaining the computer hard drives 0c cece rnnr eee e eee e eee eeee 110 Archiving and backing up experiment files 00 0 cece cece eee eee 110 Backing up the instrument SettingS 0 ccc eee eee eee eee eens 110 Decontaminating the sample block 200 0c eee eee eee e eens 111 Required materials 0c ccc ccc ee ee ence eee eee 111 Handling the sample block 0 2 0 cece eee enn net e enn e aes 111 Cleaning the sample block ccc cece eee A NR een A 112 Replacing the instrument fuses 00 unnn unnar rnnr nrnna 114 Requiredimeatertals TTT 114 Replacing the fuses 0 0 e eee aee 114 Updating the Windows operating system n n nunnan cece cece cent teen rrr 115 Updating the QuantStudio 12K Flex Software and Firmware cee ee eee eee 116 Updating the QuantStudio 12K Flex Software 0 c cece cece eee eee eee 116 Updating the QuantStudio 12K Flex Instrument firmware L e ee 116 Managing QuantStudio 12K Flex Software licenses 000 c cece cece eee ences 117 About QuantStudio 12K Flex Software license keys
280. ng the Security screen and enabling or disabling security 139 Setting the account and security policies 00 cc cece tees 139 Setting up the messaging notifications 000 cece cect tte tees 140 Managing user accountS 02 cec eect aranana 142 Creating and editing user accounts 00 eee tet ee eeeeee 142 Determining the name of the logged in user cece cee eee eee 143 Create or edita user role 2 4 bet Mena een ce ahi ee Matin ena ea eae 143 Viewing and printing a user report 0 0 0 c cece tenet eens 145 Managing auditing sie carg x cele 0 R de neva re hGe be be RRR e HR R Rees onde eee ae ne 145 Enabling disabling auditing 0 00 RET a AEUR e AER 145 Selecting objectS to audits 0 R aE 1 AE ied ae oe dee Se ea oa Ree doer ds 145 Creating audit reason settings deia enia eee aN e tenet alan 145 Generating audit reports scence eat sea dede etd R a R as a Mag dee be Agee eS 146 Displaying audit histories from the Security Settings dialog box 146 Displaying audit histories for an experiment or template eee 149 Managing electronic Signature sannan nrnna rnrn eee eee eee e ee aee 150 Enabling disabling electronic signature suinu c ccc eee eee eens 150 Configuring the meanings of the electronic signatures 2222020eeeeeeeee 150 Configuring the electronic signature rights for user roles arun nr annn n nnn 151 Selecting the
281. ng the calibration plate 1 Remove the normalization plates from the freezer then allow the plates to warm to room temperature approximately 5 minutes IMPORTANT Do not remove the normalization plates from their packaging until you are ready to run them The fluorescent dyes in the dye plates are photosensitive Prolonged exposure to light can diminish the fluorescence signal strength of the plates Note If you store the normalization plates in their original packaging and in the freezer you can use them to calibrate a QuantStudio 12K Flex System up to 3 times for 6 months after opening them Go to Performing the calibration on page 60 Before using each normalization plate vortex the plate for 5 seconds centrifuge it for 2 minutes at lt 1500 rpm then verify that the liquid in each dye plate is at the bottom of the wells If not centrifuge the plate again at a higher rpm and for a longer period of time Correct Incorrect Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time IMPORTANT The normalization plates must be well mixed and centrifuged In the Home screen of the QuantStudio 12K Flex Software click Instrument Console In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrume
282. nish the fluorescence of the dye Remove the calibration plate from its packaging Do not remove the optical film IMPORTANT Do not discard the packaging for the plate You can use the plate to calibrate a QuantStudio 12K Flex System 3 times for up to 6 months if it is stored in its sleeve Vortex and centrifuge the plate a Vortex the ROI calibration plate for 5 seconds b Centrifuge the plate for 2 minutes at lt 1500 rpm IMPORTANT The ROI calibration plate must be well mixed and centrifuged c Verify that the liquid in each well of the ROI calibration plate is at the bottom of the well If not centrifuge the plate again at greater rpm and for longer Correct Incorrect Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time 42 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks 7 ROI calibration Performing the 1 In the Home screen of the QuantStudio 12K Flex Software click Instrument calibration Console 2 In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it 3 After the QuantStudio 12K Flex Instrument is added to you
283. not calculate the threshold cycle C J Well displays the EXPFAIL flag indicating that the software cannot identify the exponential region of the amplification plot e Amplification too early e Amplification too late e Low amplification e No amplification If a well is flagged confirm the results 1 Select the flagged well s in the plate layout or well table 2 View the amplification plot Rp vs Cycle and AR vs Cycle and check for early late low or no amplification Contact Life Technologies to order a replacement TaqMan RNase P plate or array card kit Repeat the experiment making sure to properly seal and centrifuge the RNase P plate If the replacement RNase P plate or array card fails contact Life Technologies for further assistance Negative control well displays the AMPNC flag indicating that Contamination in one or more PCR reaction components Contact Life Technologies to order a replacement RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Life Technologies for further the well amplified contained in the negative control assistance well Well displays the OFFSCALE flag lt Fluorescent contaminant on 1 Perform a background calibration If you detect fluorescent contamination indicating that the fluorescence signal for one or more dyes in the well exceeds the instrument s maximum detectable range for one or more cycles th
284. ns the page describing the latest software and firmware updates for the QuantStudio 12K Flex Software and QuantStudio 12K Flex Instrument Preparing for the upgrade To update the QuantStudio 12K Flex Software prepare your computer by exporting the application libraries and backing up your experiment files To prepare for the software update 1 Back up the application libraries For each library a In the main menu of the QuantStudio 12K Flex Software select Tools gt lt library gt b When the library dialog box opens select the element s to export then click Export c In the Export dialog box click Save to archive the selected records 2 Back up all experiment files by creating a copy of the directory that you are using to store files The default directory for experiments is C Applied Biosystems QuantStudiol2KFlex User Files experiments Installing the software Install the software update according to the instructions that download with the software If you are installing the update to a computer that already contains the QuantStudio 12K Flex Software the update automatically acquires the software license from the existing installation If you are installing the QuantStudio 12K Flex Software to a computer that does not contain a previous installation you must have a license file supplied by Life Technologies If you do not have a license file obtain one as explained in Managing QuantStud
285. nt to your list before you can manage it After the QuantStudio 12K Flex Instrument is added to your list select it then click Manage Instrument In the Instrument Manager start the calibration wizard a Click Maintenance then click Normalization b In the Normalization Calibration screen click Start Calibration In the Normalization Calibration screen select the reporter passive dye combination that you want to calibrate then perform the calibration as instructed Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Normalization calibration 6 Load the calibration plate or array card into the QuantStudio 12K Flex Instrument a Verify that the normalization plate or array card matches the selection in the QuantStudio 12K Flex Software The name of the dyes contained by each consumable appears next to the barcode on the front of the plate or array card b Load the appropriate normalization plate or array card into the plate adapter Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the barcode facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned o
286. ntStudio 12K Flex Software captures a series of images of the background plate using each instrument filter The software compares the fluorescence from each well to the average for the plate A background calibration passes if the collected images for all filters are free of abnormal fluorescence About the data After the calibration you can review the calibration data in the Background tab of the Instrument Manager The Analysis Data plot left side displays the fluorescence data in all filters The Well Table tab right side displays the data collected for the current calibration The QC tab displays a summary of quality check performed by the QuantStudio 12K Flex Software on the calibration data Background fluorescence TM Fluorescence data collected by the QuantStudio 12K Flex Instrument includes a fluorescence signal inherent to the system referred to as background fluorescence Background fluorescence is a composite signal found in all spectral data that consists of fluorescence from several sources including e Background electronic signal e Contaminants in the sample block e The plastic consumable plate or array card Prepare the background calibration consumable appropriate for your instrument E Preparing the calibration plate eee ences 46 E Preparing array cards for instrument calibration lt e ssn srn ren 37 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administr
287. ntenance and Administration User Guide Chapter 6 Security Audit and Electronic Signature 6 Generating audit reports Reviewing the system configuration history The System Configuration History lists security audit and electronic signature configuration records Record type Action Corresponds to Security Settings Update Disable enable or modify security policies session timeout settings Account Settings Update Modify password settings security policies password expiration and account suspension or user name settings User Group Manager Update Create delete or modify reason for change User Role Create Create user role Delete Delete user role Update Modify user role User Account Create Create new user account Update Edit or suspend a user account Role Assignment Delete Assign a different user role to an existing user account Update Create a user account or assign a different user role to an existing account Audit Settings Update Enable or disable auditing Audit Type Update Modify audit settings FunctionManagement Update Update function management Settings Function Access Update Update function access management Manager Function Create Create function Delete Delete function Update Update function Role Permissions Create Create a user rolet Delete Delete a user role Update Modify user role p
288. o 12K Flex Real Time PCR System Maintenance and Administration User Guide 225 Results file 226 File Format Reference 7900 export format When selected as an export option the QuantStudio 12K Flex Software exports sample setup data to a stand alone file The sample setup file describes the sample configuration on the experiment consumable including sample and assay data positions and task assignments File header The file begins with a line that names the export format SDS 2 3 and the type of data contained by the file Std Results The following lines listed in the table below describe the qualities of the QuantStudio 12K Flex Instrument and several other general experiment properties Field Description Output Filename The path to the experiment file on the lt filename gt local computer hard drive PlatelD The plate identifier entered into the lt 100 character string gt barcode filed of the experiment Assay Type The type of chemistry application for Standard Curve which the experiment is designed Presence Absence Relative Standard Curve OF DDCt Quantification Run Datetime The date and time that the QuantStudio 12K Flex Instrument finished running the experiment lt date and time gt Operator The user logged into the QuantStudio lt 100 character string gt 12K Flex Software at the time the experiment was run ThermalCycleParams The thermal cycling profile for
289. o test the network connection 128 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 5 Networking 5 Collecting the required network information Collecting the required network information Obtain the following information from your network administrator e Network policy for obtaining IP addresses DHCP or static IP IMPORTANT When the QuantStudio 12K Flex System is set for DHCP APIPA is automatically enabled and the QuantStudio 12K Flex System self assigns an IP address when no address is supplied by a DHCP server e Ifthe network requires static IP addresses obtain the IP address subnet mask and gateway address for the QuantStudio 12K Flex Instrument Connecting the QuantStudio 12K Flex Instrument to the network After deciding how to connect the QuantStudio 12K Flex System to a network set up the QuantStudio 12K Flex System according to your network policies Required materials Ethernet cable with RJ45 connectors a CAT6 Ethernet cable for a 1000 Mbit s network connection or a CAT5 for 100 Mbit s connection Defining the 1 Use the Ethernet cable to connect the Ethernet port of the QuantStudio 12K Flex internet protocol Instrument to the nearest network port settings 2 Power on the QuantStudio 12K Flex Instrument 3 Use the QuantStudio 12K Flex Instrument touchscreen to configure the network settings as described in Defin
290. oftware is configured to monitor failed log in attempts you will be locked out of the software if you incorrectly enter your user name or password for a specified number of times If your user account does not have permission to perform any function in the software menu commands are grayed When your password is about to expire a message is displayed when you log in To change your password select Tools Change Password Enter your current password then enter the new password two times then click OK If the QuantStudio 12K Flex Software is configured to suspend a user account for failed logins and you enter an incorrect user name and password for more than the allowed number of times your user account is suspended and the Log In dialog box indicates that your account is inactive There are two ways to activate a suspended account e You can wait until the suspension period ends e An administrator can change the account status from Suspended to Active Note While a user is suspended another user can click Reset then log in and replace the suspended user 154 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 6 Security Audit and Electronic Signature 6 Audit Session time out If the QuantStudio 12K Flex Software is configured to time out and there is no user activity for the specified time the Log In dialog box indicates that your user session has timed
291. omputer or the monitor when lifting e Make sure that the path from where the object is to where it is being moved is clear of obstructions e Do not lift an object and twist your torso at the same time e Keep your spine in a good neutral position while lifting with your legs e Participants should coordinate lift and move intentions with each other before lifting and carrying e Instead of lifting the object from the packing box carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box Operating the instrument Ensure that anyone who operates the instrument has e Received instructions in both general safety practices for laboratories and specific safety practices for the instrument e Read and understood all applicable Safety Data Sheets SDSs Cleaning or decontaminating the instrument CAUTION Before using a cleaning or decontamination method other than those recommended by the manufacturer verify with the manufacturer that the proposed method will not damage the equipment Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 241 C Safety Instrumentation safety Physical hazard safety Electrical safety Ultraviolet light AN WARNING ULTRAVIOLET LIGHT HAZARD Looking directly at a UV light source can cause serious eye damage Never look directly at a UV light source and always prevent others from UV expo
292. on cmdlineutil ex xpgen can use the files shown below The command does not require all input files File Description assay information file Lait or aix A tab delimited or XML data file that is shipped on a CD with each TaqMan assay ordered from Life Technologies For some products assay information files are available for download from the Life Technologies website following delivery The file which contains data describing the assay can be imported into the QuantStudio 12K Flex Software for use in related experiments See Assay information file on page 207 for more information barcode file txt A user created line separated text file that contains the barcode of each consumable for which you want to create an experiment file See Barcode file format on page 207 for more information experiment A QuantStudio 12K Flex Software file that contains all information document single about a particular plate or array card consumable including metadata file eds name barcode comments plate setup well contents assay definitions run method thermal cycling protocol run results analysis protocol analysis results audit records and other plate specific data experiment A QuantStudio 12K Flex Software file used as a template to create document experiment files The file can contain plate setup well contents assay template file edt definitions run method thermal cycling
293. on doit tre effectu e par le personnel de service qualifi venant de chez Life Technologies CAUTION Moving parts Crush pinch hazard ATTENTION Pi ces en mouvement risque de pincement et ou d crasement Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 15 About This Guide Using this guide Using this guide You can use this guide to calibrate service network and administrate the Applied Biosystems QuantStudio 12K Flex Real Time PCR System This user guide contains the following information Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Describes how to maintain a QuantStudio 12K Flex System with a 96 384 well plate or array card sample block including calibration and performance verification Chapter 3 Calibrating OpenArray Plate Sample Blocks Describes how to maintain a QuantStudio 12K Flex System with an OpenArray plate sample block including calibration and performance verification Chapter 4 Maintenance Describes how to replace the user serviceable parts of the QuantStudio 12K Flex Instrument and resolve infrequent problems that can occur during normal use Chapter 5 Networking Describes how to install the QuantStudio 12K Flex System to a local area network for remote monitoring and control Chapter 6 Security Audit and Electronic Signature D
294. on plate or the sample block Common contaminants include ink residue from permanent pens powder from disposable gloves and dust To determine the source and location of the contamination 1 While viewing the background calibration data in the Analysis screen select the QC tab and review the list of through holes that failed the quality check Rotate the background plate 180 then perform the background calibration again Determine the location of the contaminated through holes again If the position s of the contaminated through hole s in step 1 and step 2 are e Identical The sample block is contaminated Decontaminate the sample block e Reversed The background plate is contaminated Discard the plate then perform the background calibration using a new background plate If the calibration fails after you replace the background plate and decontaminate the sample block a Cover a OpenArray plate with a piece of black paper b Perform the background calibration as explained in this chapter substituting the OpenArray plate covered with paper for the background plate c After the run is complete and while viewing the calibration data select all through holes in the Plate Layout tab then view the Spectral plot for the peak s If the peak associated with the contamination is e Visible The optics of your QuantStudio 12K Flex System may be contaminated Contact Life Technologies for further support
295. ond run is finished open the centrifuge and check that the fluid levels in the reservoirs of each array card have decreased by the same amount Also check for the formation of bubbles in all wells and note possible problems Correct fill Incorrect partial fill Mee MOMENO MES OMUS If necessary centrifuge the array cards for an additional minute to fill any unfilled wells Do not exceed three 1 minute runs or centrifuge the array card for longer than 1 minute at a time 11 Seal the array card s a With the carriage roller assembly of the Array AN Card Staker Sealer in the Start position place a Staker Seal Use filled array card into the fixture with the foil side up so that the fill reservoirs are the farthest away from the carriage b Press down on all four corners of the array card to ensure that it is fully seated within the fixture c Use the two alignment pins in the fixture to position the array card correctly d Seal the array card by running the carriage slowly over it Run the carriage over the array card in one direction only Do not apply downward force on the carriage as you move it forward over the card Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 39 Calibrating Multi Well Plate and Array Card Sample Blocks Preparing array cards for instrument calibration e Remove the sealed array card from the fixture and t
296. ondensation on the reaction plate Through hole displays the OUTLIERRG s Contamination 1 Decontaminate the work area and pipettors flag the C of the through hole e Improper sealing or seal leaks 2 Order and perform a replacement RNase P experiment Properly seal the deviates significantly from C values in the associated replicate group only the outlier is flagged RNase P plate If the replacement RNase P plate fails contact Life Technologies for assistance Through hole displays the THOLDFAIL flag the software cannot calculate the threshold e Amplification too early e Amplification too late e Low amplification e No amplification If a through hole is flagged confirm the results 1 Select the flagged through holef s in the plate layout or through hole table View the amplification plot R vs Cycle and AR vs Cycle and check for early late low or no amplification Order and perform a replacement RNase P experiment If the replacement RNase P plate fails contact Life Technologies for assistance Instrument malfunction Multiple possible causes Contact a local Life Technologies Field Service Office e Bunooysayqnos syoojg ajduies ajejq Aewyuedg unesqneg Chapter 3 Calibrating OpenArray Plate Sample Blocks 3 Troubleshooting Identifying Signals that exceed the limit of normal background fluorescence may indicate contamination fluorescent contaminants on the calibrati
297. onds to prevent excessive evaporation 8 Close the instrument door Note After you run the plate clean the OpenArray Accufill instrument 100 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 3 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance Sealing the 1 Remove the protective film from the top and bottom of an OpenArray Case Lid OpenArray plate IMPORTANT Remove the protective film from both sides of the lid OpenArray Lid p Protective film remove F iein L Protective film remove Notched end align with serial number 2 Grasp the OpenArray case by the top nearest the barcode using the thumb and index finger of your left hand Gently lift the case from the plate holder then load it into the OpenArray Plate Press OpenArray lid without protective film Notched end Serial number Filled OpenArray plate Barcode Align barcode here OpenArray Plate Press 3 Place the OpenArray Case Lid with protective film removed both top and bottom onto the OpenArray plate using the alignment pins of the OpenArray Plate Press for orientation 4 Actuate the OpenArray Plate Press for 10 seconds Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 101 Calibrating OpenArray Plate Sample Blocks Verifying t
298. orm the calibration as instructed When the side door opens load the ROI calibration plate or array card Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the barcode facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 6 After loading the plate or array card start the calibration a In the Setup tab select Check the box when the Uniformity Calibration plate has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 51 Calibrating Multi Well Plate and Array Card Sample Blocks Uniformity calibration 7 When the run is complete and the QuantStudio 12K Flex Software displays the Analysis screen confirm the analysis status of the calibration Select the QC tab to review the quality check summary e Analysis Status Indicates the succe
299. ort format The QuantStudio 12K Flex Software can export setup and results data from experiment files eds to tab delimited text files txt in a native QuantStudio 12K Flex System export format Data exported in the QuantStudio12KFlex export format can be opened by common spreadsheet applications such as Microsoft Excel or imported by laboratory information management system LIMS applications or databases that have been configured to parse the file format File structure The following table shows the data structure common to data exported in the QuantStudio12KFlex export format regardless of experiment type Each row represents one or more lines of data in the exported file corresponding to a common functional group The QuantStudio12KFlex export format allows the user to customize and or omit columns The columns and orders described below are the default configuration all columns in their natural order Actual files may contain fewer columns if the user modified the configuration Section Description See File header Describes the qualities of the QuantStudio 12K Flex page 210 Instrument used to run the experiment and several general experiment properties such as the date and time of the run and the dye used as the passive reference Sample setup Describes the configuration of samples on the experiment page 211 data consumable including sample location target or SNP assay properties and task assignmen
300. ou can use the regression coefficients to evaluate the quality of results from the standards See also standard curve In standard curve and relative standard curve experiments the best fit line from the standard curve Regression line formula Cr m log Qty b where m is the slope b is the y intercept and Qty is the standard quantity See also regression coefficients An action that the software performs during analysis to remove one or more wells from further analysis if a specific flag is applied to the well An experimental method to determine relative quantities This method compensates for target and endogenous control efficiency differences within each run In all experiments unknown samples and dilution series of template such as cDNA are amplified Following a run the instrument software interpolates relative quantities for each unknown sample from the appropriate dilution curve then normalizes the data for each sample or set of replicates as follows target QAvg endogenous control QAvg Expression estimation algorithm Cg which calculates a relative threshold from a fitted efficiency model for gene quantification The PCR cycle number for the threshold calculated from the modeled amplification efficiency profile A user defined biological grouping A replicate group may be a set of identical reactions in an experiment Total number of identical reactions containing identical components and identical volumes
301. ous control in a singleplex experiment the software treats separate biological samples as unpaired data when computing variability estimates of the single biological replicate Individual contributions of the separate biological samples to the single biological replicate results are observed in the Technical Replicates tab See also technical replicates In presence absence experiments a reaction that contains IPC blocking agent which blocks amplification of the internal positive control IPC In QuantStudio 12K Flex Software also the name of the task for the IPC target in wells that contain IPC blocking agent See also negative control blocked IPC wells See reference sample See reagents Method for determining relative target quantity in samples The software measures amplification of the target and of the endogenous control in samples and in a reference sample Measurements are normalized using the endogenous control The software determines the relative quantity of target in each sample by comparing normalized target quantity in each sample to normalized target quantity in the reference sample See relative threshold cycle CRT See Relative Threshold algorithm See threshold cycle CT Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 255 Glossary Cy algorithm custom dye cycle threshold cycling stage Cq data collection delta R AR diluent dilut
302. out You must enter your user name and password to access the software The administrator or another user with permission to log in to timed out sessions can click Reset then log in Audit If the QuantStudio 12K Flex Software is configured for auditing you may be prompted to specify a reason when you make certain changes in the software Depending on your QuantStudio 12K Flex Software configuration you can either select a reason from the list or enter a reason for change Electronic signature If your system is configured for electronic signature you may be required to have the experiment signed by other users before you can print a report or start a run If an item is set to require multiple signatures all approvers must sign the associated data before the action can be completed If electronic signature is enabled for experiments any of the following may apply e The Tools gt Security Sign Data menu option is enabled e You are prompted to sign as described in How the software prompts electronic signature on page 152 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 155 6 Security Audit and Electronic Signature Electronic signature 156 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Manual Instrument Operation This appendix covers Instrument touchscreen functions s 0 158
303. ow s Select Help r QuantStudio 12K Flex Software Help e Press F1 You can use the Help system to find topics of interest by e Reviewing the table of contents e Searching for a specific topic e Searching an alphabetized index Obtaining support For the latest services and support information for all locations go to www lifetechnologies com At the Life Technologies website you can e Access worldwide telephone and fax numbers to contact Life Technologies Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Life Technologies user documents SDSs certificates of analysis and other related documents e Download PDF documents e Obtain information about customer training e Download software updates and patches 252 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide AIF AIX allele allelic discrimination plot amplicon amplification amplification efficiency EFF amplification plot amplification stage Analysis Settings Library Glossary See assay information file AIF XML version of the assay information file See also assay information file AIF In a diploid organism one of two DNA sequences found at the same locus for example a particular gene but located on homologous chromosomes Two corresponding alleles may have the
304. porter The reporter dye that labels the probe for the 100 character string target assay Task The task assigned to the target in the well UNKNOWN NTC or STANDARD CT The calculated threshold cycle Cy for the target Float at the specified well Quantity e Unknown wells The calculated quantity for Float the sample at the well e Standard wells The quantity assigned to the standard at the well Quantity Mean e Unknown wells The average quantity of the Float replicate wells for the target sample e Standard wells The quantity assigned to the replicate wells for the target sample Quantity SD The standard deviation of the average quantity of Float the replicate wells for the target sample combination Ct Median The median Cy of the replicate wells for the Float specified target sample combination Ct Mean The average Cy of the replicate wells for the Float specified target sample combination Ct SD The standard deviation of the average Cy of the Float replicate wells for the specified target Automatic Ct Indicates whether the baseline was determined TRUE OF FALSE Baseline automatically true or manually false Baseline Start The first cycle used to calculate the baseline Integer Baseline End The last cycle used to calculate the baseline Integer Automatic Ct Threshold Indicates whether the threshold was determined automatically true or manually false TRUE OF FALSE Ct Threshold The C
305. r but do not go past the first stop of pipettor plunger or you may blow the solution out of the port IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port 4 Centrifuge and seal the array card as explained in Filling the calibration array cards on page 37 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 183 Calibration Consumable Preparation Creating a custom dye plate for calibration Creating a custom dye plate for calibration The Applied Biosystems QuantStudio 12K Flex Real Time PCR System can be used to run assays designed with custom dyes dyes not manufactured by Life Technologies Custom dyes must excite between 455 and 672 nm and read between 505 and 723 nm TM Before you use Before using custom dyes with the QuantStudio 12K Flex System you must custom dyes s Determine optimum dye concentration e Create a custom dye plate e Add the custom dye to the software e Perform a dye calibration Required materials Centrifuge with plate adapter e Custom dye s e Safety glasses e Powder free gloves e MicroAmp Optical 96 384 Well Reaction Plate e Optical Adhesive Cover e Pipettors and pipette tips 200 uL and 1000 uL e Tubes 2 mL and 10 mL s Deionized water Determining Note Wear powder free gloves while creating the dye plate optimum dye 1 2 Prepare and load the custom dye plate
306. r guide is written for laboratory staff who operate and maintain the TM QuantStudio 12K Flex System TM This guide assumes that your QuantStudio 12K Flex System has been installed by a Life Technologies service representative This guide also assumes that you have e Familiarity with Microsoft Windows 7 operating system e Knowledge of techniques for handling and preparing DNA samples for PCR e A general understanding of data storage file transfers and copying and pasting Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 13 About This Guide Safety information Safety information Safety alert words Safety data sheets SDSs Note For general safety information see this section and Appendix G Safety on page 237 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Four safety alert words appear in Life Technologies user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that
307. r list select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenance then click ROI b In the ROI Calibration screen click Start Calibration 5 Click Next then perform the calibration as instructed When the side door opens load the ROI calibration plate or array card Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the barcode facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 6 After loading the plate or array card start the calibration a In the Setup tab select Check the box when the ROI calibration plate has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the QuantStudio 12K Flex Instrument may pause up to 10 minutes to allow the heated cover to reach temperature 7 When the run is complete and the Analysis screen displays select each filter from the Filter Set drop down list then verify that the corresponding ROI Image displays a green circle aro
308. rds on page 140 3 Specify user password limits a Specify the passwords length limits b Specify password reuse You cannot disable the password reuse restriction c Specify the allowed characters in user passwords spaces and alphabetical numeric uppercase lowercase and special characters commas periods semicolons dashes underscores and tildes Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 139 Security Audit and Electronic Signature Configuring the system security Setting up the messaging notifications 4 In Security Policies specify Password Expiration Account Suspension and Session Timeout settings Note A session times out while a run is in progress if the time out period is exceeded and there is no other user activity In the Open Non Secure Data option select Yes or No to determine whether users can open experiments and templates that were created without security settings Click Set Up Messaging Notification Settings to specify when and how the QuantStudio 12K Flex Software notifies the administrator of certain security events For information see Setting up the messaging notifications on page 140 7 Click Apply Settings The new settings are applied to the user account the next time that the user logs in Spaces in user names and or passwords If you allow spaces in user names and or passwords be aware of the fol
309. re plots the resulting data for each spectral profile in a graph of fluorescence versus filter When the QuantStudio 12K Flex Software extracts the dye calibration data it evaluates the fluorescence signal generated by each well in terms of the collective spectra for the entire calibration plate Dye spectra are generally acceptable if they peak within the same filter as their group but diverge slightly at other wavelengths see below The QuantStudio 12K Flex Software can compensate for some differences in a spectral profile by replacing the spectra of unacceptable wells with the spectra of other wells on the reaction plate auto repairing The QuantStudio 12K Flex Software allows only a few replacements and it may reject the calibration if the spectra between neighboring wells vary significantly Note Because the wells of a calibration plate contain identical concentrations of a dye the resulting signals for the wells should be similar Variations in spectral position and peak position are caused by minor differences in the optical properties and excitation energy between the individual wells LU U 08 ence Normalized Fluoresc Normalized Fluorescence Filter Filter Acceptable spectra Unacceptable spectra Spectra peak at the same wavelength and Spectra peak at the different wavelengths do not diverge significantly Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance
310. rease from left to right and from top to bottom Contains ried b lue and g reen color values each between 0 255 The field is enclosed in double quotes with no spaces between the values Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix E File Format Reference E QuantStudio12KFlex export format Raw data The QuantStudio 12K Flex Software can export the unprocessed raw data R collected by the QuantStudio 12K Flex Instrument during the experiment run The raw data consists of fluorescence readings collected by the QuantStudio 12K Flex Instrument that have not been normalized to the passive reference The section begins with a column header followed by the raw data where each row contains the data for a single well separated by tab characters Each line of raw data consists of readings sorted by bin where each bin represents an excitation emission filter pair that was selected during experiment setup The bins are named for the corresponding filter combination according to the following convention lt excitation filter name gt lt emission filter name gt Column Description Output Well The number of the well on the consumable Integer 1 96 384 t Cycle The cycle of the run during which the Integer QuantStudio 12K Flex Instrument recorded the fluorescence lt Bin gt The raw fluorescence for the well measured by Float the QuantStudio 1
311. replicate wells of the target sample combination The lower limit of the confidence interval RQ Max The maximum relative quantity calculated for the Float replicate wells of the target sample combination The upper limit of the confidence interval Ct Mean The average Cy of the replicate wells for the Float specified target sample combination Delta Ct Mean The average AC of the replicate wells for the Float specified target sample combination Delta Ct SD The standard deviation of the AC for the replicate Float well Depending on the analysis settings this column may by replaced with Delta Ct SE the standard error of the AC Delta Delta Ct The AAC value of the replicate wells for the Float specified target sample combination Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 217 E File Format Reference QuantStudio12KFlex export format Technical replicate results The following table describes the results data exported from high resolution melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Sample Name The name of the sample contained by the well 100 character string Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well U
312. rim the fill reservoirs from the array card assembly using scissors Trim the foil array card so that the edge is even with the plastic carrier IMPORTANT Completely remove the fill reservoirs from the array card so that the edge is free of residual plastic The plastic from the fill reservoirs that extends beyond the edge of the card can prevent the array card from seating properly on the sample block and can affect amplification Correct trim Incorrect trim 6 6 e oe p d 12 Repeat step 11 to seal the remaining array cards IMPORTANT As you seal the remaining filled array cards store them in a dark place Do not expose the array cards to light until you are ready to use them The dyes in the array cards are photosensitive Prolonged exposure to light can diminish the fluorescence of the dye IMPORTANT If an array card is sealed improperly the card may leak and contaminate the sample block and or it can cause the associated calibration or RNase P experiment to fail 40 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide ROI calibration When to perform the calibration About the ROI calibration data Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks 7 ROI calibration A regions of interest ROI calibration maps the positions of the wells on the sample block of the QuantStudio 12K Flex Instrument Th
313. rom the instrument tray H WARNING PHYSICAL INJURY HAZARD During instrument operation the plates or plaque can reach 100 C Ensure the plate or plaque is at room temperature before removing Return the OpenArray Calibration Plaque to its original packaging or a clean plastic bag IMPORTANT Do not expose the OpenArray Calibration Plaque to sunlight for extended periods of time When not in use store the plaque at room temperature within the original packaging in a clean dark location IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the OpenArray Calibration Plaque remove the plate as explained in Troubleshooting on page 105 86 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 3 Calibrating OpenArray Plate Sample Blocks Uniformity calibration Uniformity calibration Required materials When to perform the calibration About the uniformity calibration IMPORTANT Perform the following procedure only if you are calibrating a QuantStudio 12K Flex System with an OpenArray plate sample block The uniformity calibration generates data that allows the QuantStudio 12K Flex Software to compensate for the physical effects of the QuantStudio 12K Flex System filters e QuantStudio 12K Flex OpenArray Calibration Plaque e Powder free gloves e Safety goggles Perform a uniformity calibratio
314. rom the rest of the plate or array card 1 While viewing the calibration data locate the wells with abnormal signal in the Plate Layout tab 2 Rotate the calibration plate or array card 180 then perform the calibration again 3 Determine the location of the contaminated wells again If the position s of the well s identified in steps 1 and 2 are e Identical The sample block is contaminated Decontaminate the sample block e Reversed The normalization plate or array card is contaminated Discard the plate or array card then perform the normalization calibration using a new normalization plate or array card 4 If the calibration fails again contact Life Technologies technical support Instrument does not eject the normalization plate The adhesive cover may have adhered the plate to the heated cover within the instrument 1 Power off the QuantStudio 12K Flex Instrument 2 Wait for 15 minutes then power on the QuantStudio 12K Flex Instrument and eject the plate 3 If the plate does not eject power off and unplug the QuantStudio 12K Flex Instrument then open the access door 4 Wearing powder free gloves reach into the QuantStudio 12K Flex Instrument and remove the plate from the heated cover then close the access door 5 Perform a background calibration to confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a
315. rotocol SMTP server to email calibration reminders Contact your systems administrator or information technology department for the following information e Network address of a SMTP server e A user name and password for the server if required for access e The Secure Sockets Layer SSL setting of the server on or off Defining the mail server settings 1 In the QuantStudio 12K Flex Software select Tools gt Preferences 2 In the Preferences dialog box select the SMTP Settings tab 3 In the SMTP Settings tab define the settings for the SMTP server e Outgoing Mail Server SMTP field Enter the network address of a Simple Mail Transfer Protocol SMTP server Optionally you can specify the transmission control protocol TCP port for the server by appending the port number to the server name separating the two using a colon For example smtp mycompany com 2023 Note If a TCP port is not specified the QuantStudio 12K Flex Software uses the default port number 25 s Encryption Required Select if the mail server has SSL enabled e Authentication Required Select if the mail server requires a user name and password e User Name and Password fields If the mail server requires authentication enter the user name provided by your systems administrator 4 Click OK 134 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 5 Networ
316. rt format sev chet ini shine baad E A E aides 228 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 199 E File Format Reference Import formats and file specifications Import formats and file specifications The QuantStudio 12K Flex Software supports several import file formats that can be used to automate experiment creation and assay and sample data import The files can be used with the command line application see page 189 or the QuantStudio 12K Flex Software application programming interface API to integrate the QuantStudio 12K Flex System into a laboratory information management system LIMS For a detailed explanation of the API or for information on integrating the QuantStudio 12K Flex System into a laboratory workflow see the Applied Biosystems QuantStudio 12K Flex Real Time PCR System Automation Guide Part no 4470693 Note The file specifications listed in this appendix are subject to change For updated information review the QuantStudio 12K Flex Software Release Notes found at C Program Files Applied Biosystems QuantStudio12KFlex docs README html About the import f File format Description See file formats E Plate setup file A user created tab delimited text file that describes the page 201 LH layout of a consumable for an experiment to be run on the QuantStudio 12K Flex System The file defines the arrangement of assay
317. rt the QuantStudio 12K Flex Software 132 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 5 Networking 5 Monitoring a QuantStudio 12K Flex Instrument Uploading or Note The QuantStudio 12K Flex Instrument can store up to 100 gene expression downlo ading an experiments Before sending an experiment confirm that the instrument contains experiment or sufficient storage space template 1 In the Home screen of the QuantStudio 12K Flex Software click Instrument Console 2 In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it 3 After the QuantStudio 12K Flex Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager click Manage Files then click File Manager 5 In the File Manager screen transfer the file s To upload a file to the QuantStudio 12K Flex Instrument a In the Folders field select the folder to which you want to upload the file To create a new folder click Create then enter a name for the new folder b Click Upload select the experiment or template file to send to the QuantStudio 12K Flex Instrument then click Open To download a file from the QuantStudio 12K Flex Instrument a In the Fold
318. rvoltage rating The QuantStudio 12K Flex System has an installation overvoltage category of II and is classified as portable equipment 242 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Bar code scanner laser safety Workstation safety Appendix G Safety G Instrumentation safety Laser classification The bar code scanners included with the QuantStudio 12K Flex Instrument are categorized as Class 2 II lasers Laser safety requirements Class 2 II lasers are low power visible light lasers that can damage the eyes Never look directly into the laser beam The scanner is designed to prevent human access to harmful levels of laser light during normal operation user maintenance or during prescribed service operations WARNING LASER HAZARD Class 2 II lasers can cause damage to eyes Avoid looking into a Class 2 II laser beam or pointing a Class 2 II laser beam into another person s eyes Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions CAUTION MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions
319. s Provide USB communication with the QuantStudio 12K Flex Instrument Can be used to transfer data to and from the instrument and to update the firmware Note If multiple USB drives are plugged into the QuantStudio 12K Flex Instrument the instrument mounts only the first drive that is installed regardless of the USB port used B Instrument touchscreen Provides access to the QuantStudio 12K Flex Instrument functions Can be used to run experiments transfer data and operate the instrument functions without the use of the computer C Access door Provides access to the QuantStudio 12K Flex Instrument LED the heated cover and the sample block LED Illuminates the reaction plate or array card during a run Heated cover Covers the plate or array card during a run to prevent condensation and leakage through the consumable cover Sample block Heats the plate or array card during a run Side door Opens to allow extension of the tray arm IO 7 Plate adapter Secures plates or array cards to the tray arm Tray arm Conveys plates or array cards to and from the sample block in the interior of the QuantStudio 12K Flex Instrument 24 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Rear view Chapter 1 Getting Started 1 QuantStudio 12K Flex System hardware Ethernet
320. s provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste e Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS e Handle chemical wastes in a fume hood e After emptying a waste container seal it with the cap provided e Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory e Ensure the health and safety of all personnel in your laboratory e
321. s and file specificationS ccc eee eee e eee aes 200 About the import file formats 000 cece eee teenies 200 CONVENTIONS EaR aE se cera eben ee ss Ging eis dodges eu Be seed woes 200 Plate setup file format 0 0 00 cece ccc e eee eee 201 PPULGISEPUICTUN Os 2 2 255 E Seta cyte at cS kcal Mi hia E H E 201 Plate setup file header 000 cece eee eens 201 Plate setup file body 0c e eee eee eens 202 Plate setup data columns 0c c eect e eee e eens 203 Example Sei Ache E coins ates Peet newt ot Ad ele Yaa ah one aad A Ae ae ay 204 Sample file formate ma Re Ya ZZ Weare ce a ee HATE EE TEE EAR N Reuters Bedard 206 Rile structure eaae Wagan a aase ks a R a a A AE E hee sere eae 206 Example file Aiari i ag N YR ee AE valved Aa ERRENA RR hoa AEP Ya 206 Barcodetile format lt 249 e 727 Re 72 R 9 ward el Rae E a my eel ded 207 Bile StrulCtutee rninha aaa R eaten eh es eet oars hres rendre Casta aise Rca aie Steno te Vaca 207 XM PLS TIL nm ohh i Ae ete ge a tte es cok e Ae eee aa aay Reine ON 207 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 9 Contents Assay information file e e e e K K eee eee en rrer renaren 207 Export formats and file specifications 0c eee eee eee 208 EXxXportformats since ue aed one Ue aed On eee ate one eae et ieee 208 QuantStudio12KFlex export format e cece eee nett raran e aes 209 Fil StrUctUre es tet te ea
322. s and samples on the consumable and provides other experiment data such as the thermal profile and data collection settings Sample file txt A user created tab delimited text file containing sample page 206 data that can be imported into the QuantStudio 12K Flex Software for use in related experiments Assay information A tab delimited or XML data file that is shipped on a CD page 207 file Lair or aix with each TaqMan assay ordered from Life Technologies The file which contains data describing the assay can be imported into the QuantStudio 12K Flex Software for use in related experiments Barcode file txt A user created text file containing the barcodes of page 207 consumables for which you want to create experiment files using the command line utility Conventions The following conventions are used in the rest of this section e normal Normal text must be entered exactly as it appears s lt italic gt Italicized text between brackets must be substituted with custom values e required text Text appearing between brackets is required information All information inside the brackets must be present for the QuantStudio 12K Flex Software to import it e optional text Text appearing between braces is optional e Unless noted otherwise separate all fields in a row using a tab character U 0009 e Unless noted otherwise end all rows using a carriage return character U 00
323. s at the locations shown labels on below instruments an N Nng g 240 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide General instrument safety Appendix G Safety G Instrumentation safety WARNING PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Life Technologies may result in personal injury or damage to the instrument Moving and lifting the instrument CAUTION PHYSICAL INJURY HAZARD The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide If you decide to lift or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons Moving and lifting stand alone computers and monitors WARNING Do not attempt to lift or move the computer or the monitor without the assistance of others Depending on the weight of the computer and or the monitor moving them may require two or more people Things to consider before lifting the computer and or the monitor e Make sure that you have a secure comfortable grip on the c
324. s lower background signals resulting in improved precision in quantification The minor groove binder MGB increases the melting temperature Tm of the probe without increasing its length allowing for the design of shorter probes Type of calibration in which the software collects data from the normalization standards then stores it in a normalization calibration file This file is used in comparisons of data from multiple instruments within a study Either the Cy Avg of the target gene minus the Cy Avg of the endogenous control Comparative Cy experiments or the Q Avg of the target divided by the Q Avg of the endogenous control Relative Standard Curve experiments The relative standard curve equivalent of the AC mean value found in Comparative Cy experiments computed as the geometric mean The relative standard curve equivalent of the AC SE value found in Comparative Cy experiments computed as the geometric standard error of the mean Fluorescence signal from the reporter dye normalized to the fluorescence signal of the passive reference dye usually ROX dye on Life Technologies instruments An action that you perform before reanalysis to omit one or more wells from analysis Because no algorithms are applied to omitted wells omitted wells contain no results You can add wells back in to the analysis no information is permanently discarded A measurement such as a Cy that deviates significantly from the measurement of
325. s not validate the barcodes Example file HA996346102 IB894812348 DD834814679 EK209825848 AF092387348 FF225676243 Assay information file The QuantStudio 12K Flex Software command line application can import data for Life Technologies assays from assay information files aif which is shipped on a CD with each assay order The aif contains technical details about all assays in the shipment It includes information about assay concentrations reporters and quenchers used part and lot numbers and assay vial and plate ID numbers The file name includes the number from the barcode on the plate Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 207 E File Format Reference Export formats and file specifications Export formats and file specifications Export formats This section describes the export formats supported by the QuantStudio 12K Flex Software The information provided in this appendix is intended for users who want to integrate the QuantStudio 12K Flex Software with third party applications including downstream analysis software and laboratory information management system LIMS tools Note The file specifications listed in this appendix are subject to change For updated information review the QuantStudio 12K Flex Software Release Notes found at C Program Files Applied Biosystems QuantStudio12KFlex docs README html The QuantStudio
326. s of the step For cycling stages a step is also defined by the AutoDelta status PCR reaction components that consist of two primers designed to amplify the target and SYBR Green dye to facilitate detection of the PCR product 266 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide system dye TaqMan reagents target target color Target Library task technical replicates temperature plot template template file thermal profile Glossary Dye supplied by Life Technologies and precalibrated on the QuantStudio 12K Flex System Before you use system dyes in your experiments make sure the system dye calibration is current in the Instrument Console The system dyes are e FAM dye s JOE dye s ROX dye e NED dye e SYBR Green dye e TAMRA dye s VIC dye PCR reaction components that consist of primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target The nucleic acid sequence to amplify and detect In the software a color assigned to a target to identify the target in the plate layout and analysis plots In the software an editable collection of targets to use in experiments Targets in the library contain the target name reporter quencher and target color The targets in the library may also contain comments about the target In the software the type of reaction performed in the we
327. ser Guide 99 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance 2 Verify each of the following conditions and select each check box e Tips are configured e Waste bin is empty e OpenArray plate is in the plate holder Select the state of the tip boxes by clicking on the sections Tip Box 1 Tip Box 2 Verify T The tips are configured as shown above T The Waste Bin is empty T The OpenArray Plate is in the Plate Holder T Remove foil from the highlighted section of the Sample Plate Close the Instrument Door lt Back Load Cancel Note The software will not continue until you select all the check boxes 3 With forceps peel off the foil covering the area of the sample plate containing the samples to be loaded on the OpenArray plate 4 Select Remove foil from the highlighted section of the Sample Plate 5 Close the instrument door 6 Click Load Note If the number of OpenArray plates in the instrument differs from the number that is entered in the Setup Load Information window an error message instructs you to remove any extra plates Correct the error and continue 7 When the Remove OpenArray Plate window appears open the instrument door carefully remove the indicated OpenArray plate then immediately seal the plate as explained in Sealing the OpenArray plate on page 101 IMPORTANT Once an OpenArray plate has been filled seal it within 90 sec
328. setup column header and body can appear in any order IMPORTANT Observe the following guidelines when creating a plate setup file e Do not insert blank lines between the sample setup header and the column header e Do not use illegal characters including backslash tab asterisk hard return soft return brackets or or comma Sample setup header The header contains the label that defines the beginning of the sample setup data Example Sample Setup Sample setup column header The column header contains the headings that define the positions of the data columns in the sample setup body The headings are separated by tab characters See Plate setup data columns on page 203 for a list of the data column headers Example Well Sample Name Sample Color Biogroup Name Biogroup Color Target Name Sample setup body Contains the sample setup data where each row defines the contents of a single well on the consumable including the well contents sample target or SNP assay added to the well task assignments and comments If a well contains multiple assays multiplex PCR the data for the additional assays are defined on separate lines by repeating the well designation See Plate setup data columns on page 203 for a list of the data column headers Note The sample setup data rows can occur in any order Example Well Sample Name Sample Color Biogroup Name Biogroup Color Target Name Or
329. sistance RNase P plate verification run failed Defective plate seal CAUTION PHYSICAL INJURY HAZARD During instrument operation the sample block can reach 100 C Allow the plate to reach room temperature before removing Unload the OpenArray plate from the QuantStudio 12K Flex Instrument Hold the OpenArray plate up to a light source and verify that the plate contains fluid and that bubbles are not present Order and perform a replacement RNase P experiment If the replacement OpenArray plate fails contact Life Technologies for further assistance Instrument does not eject the RNase P plate Adhesive cover may have adhered the plate to the heated cover within the instrument Power off the QuantStudio 12K Flex Instrument Wait for 15 minutes then power on the QuantStudio 12K Flex Instrument and eject the plate Ifthe plate does not eject power off and unplug the QuantStudio 12K Flex Instrument then open the access door Wearing powder free gloves reach into the instrument and remove the plate from the heated cover then close the access door Through hole displays the NOSIGNAL flag the through hole produced very low or no fluorescence signal Missing reaction mix resulting from pipetting error If a through hole is flagged confirm the results 1 2 Consider omitting the through hole from the analysis Note the location for each flagged through hole
330. so change the plate adapter to match the new consumable format 1 Power off and unplug the QuantStudio 12K Flex Instrument then allow it to cool for 15 minutes 2 Open the access door Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 119 120 Maintenance Replacing the sample block Firmly press down on the handle of the sample block then remove it from the QuantStudio 12K Flex Instrument Place the sample block on a clean dry surface Install the new sample block into the QuantStudio 12K Flex Instrument IMPORTANT After installing the sample block confirm that the indicator on the left side of the sample block is positioned behind the red line on the instrument rail If the indicator is forward of the red line push the sample block into the QuantStudio 12K Flex Instrument until it is seated correctly If you are installing a sample block of a different consumable format replace the heated cover and plate adapter if necessary to match the new consumable format IMPORTANT If you are installing a sample block of a different format you must also change the plate adapter to match the new consumable format Close the access door IMPORTANT Confirm that the access door is completely closed The QuantStudio 12K Flex Software displays an error message if the door is not completely closed and latched or if the sample block is not seat
331. ss of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or the data it collected is unusable Note A calibration can fail if wells produce spectra that deviate significantly from the other wells of the plate or if all wells produce abnormally low spectra Abnormal spectra can indicate the presence of fluorescent contamination on the plate or array card or sample block e QC Status Indicates the quality of the calibration data where passed indicates that all wells produced data that passed the quality check and failed indicates that one or more wells produced spectra that deviate significantly from the other wells on the plate Analysis ac Action status status Passed Passed Click Next then remove the plate or array card when the QuantStudio 12K Flex Instrument ejects the tray arm Passed Failed Troubleshoot the failed calibration as described in Table 4 Troubleshooting uniformity calibrations on page 74 Note You can accept a calibration that passes the Analysis Failed Failed Status check but fails the QC Status check We recommend using calibrations that yield passing results for both status reports WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing IMPORTANT If the QuantStudio 12K Flex Instrument do
332. status reports WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the plate remove the plate according to Table 5 Troubleshooting dye calibrations on page 75 10 Discard or store the consumable Consumable Action Array card Discard the array card Plate Store the dye calibration plate in the freezer in its packaging sleeve IMPORTANT Do not discard the calibration plate If the plate is stored frozen in its packaging sleeve you can use it to calibrate a QuantStudio 12K Flex System 3 times for up to 6 months after you open it 11 Repeat steps 4 to 10 as needed to calibrate the QuantStudio 12K Flex System for the remaining dyes in the chemistries that you are running 58 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Normalization calibration Normalization calibration When to perform the calibration About the normalization calibration data Preparing the calibration plate or array card Preparing the normalization plates During the normalization calibration the QuantStudio 12K Flex System e Collects data from the normalization standards e Stores the information for
333. strument Temperature Plot Observe the restrictions to mDNS and Autodiscovery The QuantStudio 12K Flex System supports mDNS but only when the QuantStudio 12K Flex Instrument and computer share a direct network connection and are within the same subnet Consequently network computers that are separated from the QuantStudio 12K Flex System by a router hub or another network device may not be able to access the QuantStudio 12K Flex Instrument by its host name Confirm the uniqueness of the instrument name The QuantStudio 12K Flex Instrument does support name resolution but the instrument name must be unique within the subnet The QuantStudio 12K Flex Software can automatically discover QuantStudio 12K Flex Instruments on the link local network that are configured for Autodiscovery see Defining the network settings on page 170 Note The QuantStudio 12K Flex System does not test the uniqueness of the instrument name when it is set Name QuantStudio 12K Flex Instruments using lower case letters When you define the QuantStudio 12K Flex Instrument settings see Defining the instrument settings on page 168 enter the instrument name using lower case letters only Network setup workflow ES YN Collect the required network information Connect the QuantStudio 12K Flex Instrument to the network Connect the computer to the network Monitor the QuantStudio 12K Flex Instrument t
334. sure Follow the manufacturer s recommendations for appropriate protective eyewear and clothing Moving parts WARNING PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing the instrument WARNING ELECTRICAL SHOCK HAZARD Severe electrical shock can result from operating the QuantStudio 12K Flex Instrument without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument Fuses WARNING FIRE HAZARD Improper fuses or high voltage supply can damage the instrument wiring system and cause a fire Before turning on the instrument verify that the fuses are properly installed and that the instrument voltage matches the power supply in your laboratory WARNING FIRE HAZARD For continued protection against the risk of fire replace fuses only with fuses of the type and rating specified for the instrument Power WARNING ELECTRICAL HAZARD Grounding circuit continuity is required for the safe operation of equipment Never operate equipment with the grounding conductor disconnected WARNING ELECTRICAL HAZARD Use properly configured and approved line cords for the voltage supply in your facility WARNING ELECTRICAL HAZARD Plug the system into a properly grounded receptacle with adequate current capacity Ove
335. t a aa mye et ten ate cot Bel ty aa alan Seige Pee 209 Bile header 2s ones ve vad esis added eh seed Seeds eee oan Meee 210 Sample setup data 2 2c cece eee eet eect tte ett eeeeeee 211 Raw data ss poies ATR AR aa TSK EAA ARTE A EE A ea eid eens RATRE he 213 Amplification datace tases aac tite at noden A aide ne Ase et htt eet ioe are oe 214 Multicomponent datal 2 00 4 teens eee eee cab pea oe een alae 2 215 Results datas erase 2 L gta tae hee ee tee cae tt oe Ee ite te SiS ee ee eA ee eee Pee 215 7900 export format 2 ce 2 vag ye Sen Peed ven E a Heee vine vee eee eevee ee pees eee eae Peds 223 Exportable tiles 5 12 cegees omega dee von A ATRE ene T E RTA eed N ARR eee 223 DOCU TLS eae sae kere ales TTT 223 File N ader isos 238 eel ead eel OA cet So Net aM te gt ah hae dol 223 Assay det ctor data micri annaia aeda e leads cud stig s ade EA dR AR DED 224 Well data i c 0 os5 fae eid ep ey oda e E a e tee ede ee oe ee a pe oe hee 224 Multicomponent Tile 2 2 0254 sc cene st na debs ence fobs K RAN K RARE cee ha 225 Results He 0 6 T a de E Y ee hee evra ae wales Gabe pete ade ke Ca de R NR L g debate 226 Standard Curve Relative Standard Curve and Comparative Cy experiments 227 Genotyping experiments n nsanu nann cece cece cette eet tet eane 228 ROMEexport formats i6 t 4545 ee eee heh eel ee cel bh hel cE te oe 228 For more information e c2004 sae usher gta yee eg See yd Ming Seat Wee eo ean 228 APPENDIX F Parts
336. t cycle used to calculate the baseline for Float Baseline Start allele 2 Allele2 The last cycle used to calculate the baseline for Float Baseline End allele 2 Custom1 The contents of the custom text fields found inthe 1024 characterstring Custom Results table of the experiment per field Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 219 File Format Reference QuantStudio12KFlex export format Melting curve The following table describes the results data exported from melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 character string Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN Or NTC Reporter The reporter dye that labels the probe for the target assay 100 character string Quencher The quencher dye that labels the probe for the target assay 100 character string CT The calculated threshold cycle C7 for the target at the specified wel
337. t to calibrate a QuantStudio 12K Flex System 3 times for up to 6 months after you open it 9 In the Background Calibration screen click Finish to complete the calibration then click Yes when prompted to save the results 48 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Uniformity calibration Uniformity calibration The uniformity calibration generates data that allows the QuantStudio 12K Flex Software to compensate for the physical effects of the QuantStudio 12K Flex System filters When to perform Perform a uniformity calibration every year or as often as necessary depending on the calibration instrument use About the During the uniformity calibration the QuantStudio 12K Flex Software captures a uniformity series of images of the ROI plate using each instrument filter After the calibration you calibration data can review the data in the Uniformity tab of the Instrument Manager The Analysis Data plot left side displays the fluorescence data in all filters The Well Table tab right side displays the data collected for the current calibration in all well positions The QC tab displays a summary of quality check performed by the QuantStudio 12K Flex Software on the calibration data Preparing the If you have an ROI plate or array card from a recent ROI calibration go to step b o
338. t you want to receive email notifications Separate multiple email addresses with commas 4 Click Apply to change the notification settings Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 135 5 Networking Monitoring a QuantStudio 12K Flex Instrument 136 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Security Audit and Electronic Signature This chapter covers S Administrators Overview e 138 E Configuring the system security 0 ee eens 139 M Managing user accounts s s s resser eens 142 M Managing auditing ERER TRE R eee R 0 es 145 R Generating audit report e e nents 146 S Managing electronic signature 2 0 6 cee eee 150 E Generating electronic signature reports eee eee 152 m Exporting and importing settings e rre rrrr rererere 153 ME E e a L 154 BB Seca 2 fata tin ch ah HD a hbase Ghee aed Suh I heath he Aas E E 154 R Au aed RAE KE EET RAR Te E Ais Rs AEE Sa DAA La EE ees 155 M Electronic signature ERa THESE NER AHE R ER NTT 155 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 137 Administrators overview 6 Security Audit and Electronic Signature Administrators overview Example applications 138 IMPORTANT The Security Audit and Electronic Signature SAE module
339. taminating the heated heated cover cover handle the assembly as shown below After the assembly has been removed from the QuantStudio 12K Flex Instrument place the heated cover on a clean dry surface or in its shipping container Replacing the Note Confirm that the replacement heated cover supports the consumable format that heated cover you want to use Some heated covers support more than one consumable type 1 Power off and unplug the QuantStudio 12K Flex System then allow it to cool for 15 minutes 2 Open the access door 3 Unlock the heated cover by pinching the handle together then pull the assembly from the QuantStudio 12K Flex Instrument and place it on a clean dry surface Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 121 122 Maintenance Replacing the heated cover Install the new heated cover into the QuantStudio 12K Flex Instrument IMPORTANT When the heated cover is seated correctly the arrows on the front handle align as shown below If the arrows do not align push the heated cover further into the QuantStudio 12K Flex Instrument until the handle locks into place Correct Incorrect If you are installing a heated cover of a different consumable format replace the sample block and plate adapter if necessary IMPORTANT If you are installing a heated cover of a different format you must also change the sa
340. tarted or a self test is intentionally run The System Self Test utility is in the Instrument drop down menu in the OpenArray Accufill application Click Setup amp Load Setup Load Information Loading information Specify the type of load you want to perform Enter Plate and OpenArray Plate barcodes by scanning them typing them or selecting the associated file Force Single Sample Loading Sample Plate TestSamplePlate Plate Holder Position TST01 Browse Plate Holder Position2 TsTo2 Browse Use Sample Integration Plate Holder Position 3 Plate Holder Position 4 Browse Select Samples to Load Click the section on the sample plate from which the first sample will be extracted The image on the right shows how the sample will be applied to the OpenArray Plates Plate Holder Position 1 TST01 ate Halder Position 2 IRIN NUT TST02 late Holder Position 3 IRH 5 ate Holder Position 4 IWIN Next Cancel 2 Open the enclosure door of the OpenArray Accufill instrument by grasping and lifting up the door handle Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Loading the OpenArray plate Chapter 3 Calibrating OpenArray Plate Sample Blocks Verifying the instrument performance Inthe Setup Load Information window enter or scan the barcode of your TaqMan RNase P Op
341. ted from the slope knowing the error allows a CI for the amplification efficiency to be calculated Refers to the entire process of performing a run including setup run and analysis You can perform the following types of experiments e Quantification Standard curve e Quantification Relative standard curve e Quantification Comparative Cr AAC Melt Curve e Genotyping e Presence absence The Life Technologies name for the electronic records that comprise all information about a particular plate or array card consumable including metadata name bar code comments plate setup well contents assay definitions run method thermal cycling protocol run results analysis protocol analysis results audit records and other plate specific data Experiment documents have the suffixes eds experiment document single edt template and edm multiple Entered during experiment setup the name that is used to identify the experiment A software feature that allows you to set up an experiment according to your experiment design Experiment Setup provides you with maximum flexibility in the design and setup of your experiment The type of experiment to perform e Standard curve e Comparative Cy AACT e Relative standard curve e Genotyping e Presence absence e Melt curve The experiment type that you select affects setup run and analysis A software feature that allows you to export experiment setup files experiment
342. tem Software QuantStudio 12K Flex System Software Computer requirements Software installation 30 The QuantStudio 12K Flex System includes a suite of software applications that can be used to calibrate run automate and integrate the QuantStudio 12K Flex System into a laboratory workflow The basic installation of the QuantStudio 12K Flex Software contains the components described below however additional software may be available for the QuantStudio 12K Flex System Visit the QuantStudio 12K Flex System website for a complete list of compatible software www lifetechnologies com quantstudio12K flex Note Visit the QuantStudio 12K Flex System website for updates and patches for the QuantStudio 12K Flex Software and QuantStudio 12K Flex Instrument Firmware The requirements for the computer used to operate the QuantStudio 12K Flex Instrument can vary depending on the version of the QuantStudio 12K Flex Software that you are running To determine the computer requirements for your QuantStudio 12K Flex System check the QuantStudio 12K Flex Software release notes at the following location C Applied Biosystems QuantStudiol2KFlex README html The default installation of the QuantStudio 12K Flex System partitions the computer hard drive to create the logical drives shown below Drive Software Description C Microsoft Windows OSF Operating system files Qu
343. tenance and Administration User Guide 91 Calibrating OpenArray Plate Sample Blocks Dye calibration Perform the filled reading c Load case 1 into the position closest to the QuantStudio 12K Flex Instrument followed by the remaining cases in sequence as shown in the following figure Plugs IMPORTANT Confirm that the OpenArray Calibration Cases are positioned so that the plugs are oriented away from the A1 position as shown IMPORTANT The instrument should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained After loading the OpenArray cases start the calibration a In the Dye Calibration screen select Check the box when the dye calibration cases have been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the QuantStudio 12K Flex Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature IMPORTANT Wear powder free gloves while preparing the OpenArray Calibration Cases When the instrument door opens and you are prompted to perform the filled reading load the OpenArray Calibration Cases with OpenArray FAM Dye Solution 1 Attach a syringe tip to the syringe then place the assembly on a clean surface
344. ter that you have provided install the QuantStudio QuantStudio 12K 12K Flex Software using the Applied Biosystems QuantStudio 12K Flex Flex Software Software CD Note You must install the QuantStudio 12K Flex Software to monitor the QuantStudio 12K Flex System over the network 2 Optional Install protective software to the computer Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 131 5 Networking Monitoring a QuantStudio 12K Flex Instrument Monitoring a QuantStudio 12K Flex Instrument After connecting the QuantStudio 12K Flex System and a computer to the network you can enable remote monitoring in the QuantStudio 12K Flex Software to observe the instrument status remotely About remote When the QuantStudio 12K Flex System is connected to the network any computer monito ring on the network that is running the QuantStudio 12K Flex Software can S Monitoring the status of an instrument during a run see below S Uploading or downloading an experiment or template 133 m Enabling or changing the calibration reminders 0 000000 rnn 134 Guidelines for remote monitoring To ensure optimal performance of the remote monitoring feature observe the following guidelines s The QuantStudio 12K Flex Software can monitor up to 15 instruments s We do not recommend that a QuantStudio 12K Flex Instrument
345. the other replicates for that same sample Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide passive reference plate layout plate setup file point positive control post PCR read pre PCR read primer mix primer probe mix pure dye quantification cycle G Glossary A dye that produces fluorescence signal independent of PCR amplification and that is added to each reaction at a constant concentration Because the passive reference signal should be consistent across all wells it is used to normalize the reporter dye signal to account for non PCR related fluorescence fluctuations caused by minor well to well differences in volume Normalization to the passive reference signal generally results in data with noticeably high precision among technical replicates An illustration of the grid of wells and assigned content in the reaction plate The number of rows and columns in the grid depends on the sample block that you use In the software you can use the plate layout as a selection tool to assign well contents to view well assignments and to view results The plate layout can be printed included in a report exported and saved as a slide for a presentation A file txt csv xml or sds that contains setup information such as the well number sample name sample color target name dyes and other reaction plate contents One standard in a standard curve Th
346. the 96 384 well or experiment array card The QuantStudio 12K Flex Software can export the results data from an analyzed experiment file The format and content of the results data depends on the experiment type and the analysis settings The section begins with a column header followed by the results data where each row contains the data for a single well separated by tab characters If a well contains more than one assay target the QuantStudio 12K Flex Software lists the data for each additional assay on separate rows repeating the well number and sample information This section describes the following results data formats S Standard Curve Relative Standard Curve and Comparative CT experiments 227 m Genotyping experiments Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Standard Curve Relative Standard Curve and Comparative Cy experiments Appendix E File Format Reference 7900 export format The following table describes the results data exported from standard curve relative standard curve and comparative Cy experiments Column Description Output Well The number of the well on the consumable Integer 1 96 384 Sample Name The name of the sample contained by the well 100 character string Detector The name of the target assay added to the well 100 character string Name Re
347. the associated sample RGB lt r gt lt g gt lt b gt 8 SNP Assay The name of the SNP assay applied to the well 100 character string Name If the well contains multiple assays the data for each SNP assay are exported in an additional row SNP Assay The RGB color of the associated SNP assay RGB lt P gt lt g gt lt b gt Color Task The task assignment of the SNP assay at the UNKNOWN Or NTC well Allele1 Name The name of the first allele for the associated SNP assay 100 character string Allele1 Color The RGB color of the first allele for the associated SNP assay RGB lt r gt lt g gt lt b gt 8 Allele1 The reporter dye that labels the probe for the 100 character string Reporter first allele Allele1 The quencher dye that labels the probe for the 100 character string Quencher first allele Allele2 Name The name of the second allele for the associated SNP assay 100 character string Allele2 Color The RGB color of the second allele for the associated SNP assay RGB lt r gt lt g gt lt b gt Allele2 The reporter dye that labels the probe for the 100 character string Reporter second allele Allele2 The quencher dye that labels the probe for the 100 character string Quencher second allele Comments Additional text that describes the well 1024 character string Well numbers start at 1 for well A1 upper left corner and inc
348. ting curvet Post PCR Genotyping The QuantStudio 12K Flex Instrument collects endpoint data e Before the PCR For presence absence experiments data collection before the PCR is optional but recommended e Optional During the PCR The QuantStudio 12K Flex Instrument can collect data during the run real time collecting real time data during the run can be helpful for troubleshooting endpoint results e After the PCR Presence absencet T Not available for OpenArray experiments 18 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Instrument filters and supported dyes Chapter 1 Getting Started About the QuantStudio 12K Flex System Regardless of the run type a data collection point or read consists of three phases 1 Excitation The QuantStudio 12K Flex Instrument illuminates all wells of the reaction plate within the instrument exciting the fluorophores in each reaction 2 Emission The QuantStudio 12K Flex Instrument optics collect the residual fluorescence emitted from the wells of the reaction plate The resulting image collected by the device consists only of light that corresponds to the range of emission wavelengths 3 Collection The QuantStudio 12K Flex Instrument assembles a digital representation of the residual fluorescence collected over a fixed time interval The QuantStudio 12K Flex Software s
349. tional Specifies the path and name lt filepath gt of the setup file that the software uses to create new files Example s C setupfiles setupfile txt t lt filepath gt Required Specifies the path and name lt filepath gt of the QuantStudio 12K Flex Software template file that the software uses to create new files Example t C Applied Biosystems QuantStudio 12K Flex Software User Files experiments templatefile edt Optional Configures the software to operate in verbose mode where the software displays each operation as it is performed Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 195 D Command line Software Operation Command syntax and arguments Results export The command used to export the results from experiment files uses the following syntax cmdlineutil exe export parameters The following table lists the acceptable parameters that can be included in any order See Examples on page 197 for and examples of the experiment export command IMPORTANT Enclose file paths in double quotes to allow spaces in the string e lt dirpath gt Required Specifies the path to the directory lt dirpath gt that contains the experiment files eds for which the software exports data Example e C Applied Biosystems QuantStudio 12K Flex Software User Files experiments f lt option gt Requ
350. to prepare your site for installation of the QuantStudio 12K Flex Instrument Applied Biosystems 4442662 Explains how to install and maintain the ViiA 7 Real Time PCR Instrument nTM ee hie Intended for laboratory staff responsible for the installation and maintenance of et the ViiA 7 Real Time PCR Instrument Installation Guide QuantStudio 12K Flex NA Explains how to use the QuantStudio 12K Flex Software to Software Help e Set up run and analyze experiments e Monitor a networked QuantStudio 12K Flex Instrument e Calibrate the QuantStudio 12K Flex Instrument e Verify the performance of QuantStudio 12K Flex Instrument with an RNase P run e Intended for Laboratory staff and principal investigators who perform experiments using the QuantStudio 12K Flex System Laboratory staff responsible for the installation and maintenance of the QuantStudio 12K Flex Instrument Note For additional documentation see Obtaining support on page 252 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 251 Documentation and Support Obtaining information from the Help system Obtaining information from the Help system The QuantStudio 12K Flex System has a Help system that describes how to use each feature of the user interface Access the Help system by doing one of the following s Click in the QuantStudio 12K Flex Software wind
351. tores the raw fluorescent image for analysis After a run the QuantStudio 12K Flex Software uses calibration data to determine the location and intensity of the fluorescent signals in each read the dye associated with each fluorescent signal and the significance of the signal System dyes The Applied Biosystems QuantStudio 12K Flex Real Time PCR System features a six color filter set that supports all Life Technologies dyes The following figure shows the emission spectrum for each dye and the filter at which each dye is read Nies See eee I l I 500 Wavelength nm E E R Emission pe Spectra ee il Ee Dyes 520 nm 550 nm 580 nm 610 nm FAM VIC NED ROX SYBR Green TAMRA y Filter wavelength nm t peg Color Supported dyes Se Excitation Emission x1 m1 Blue 470 15 520 15 FAM and SYBR Green dyes x2 m2 Green 520 10 558 12 VIC JOE TET and HEX dyes x3 m3 Yellow 549 5 10 586 5 10 NED and TAMRA dyes x4 m4 Orange 580 10 623 14 ROX dye x5 m5 Red 640 10 682 14 LIZ dye x6 m6 Deep red 662 10 71112 None T The central wavelengths are the optimized wavelengths No Life Technologies supported dye currently available Custom dyes The Applied Biosystems QuantStudio 12K Flex Real Time PCR System can run assays designed with custom dyes dyes not supplied by Life Technologies that are excited between 455 672 nm
352. tration User Guide Appendix A Manual Instrument Operation Administering the instrument from the touchscreen Administering the instrument from the touchscreen The touchscreen provides access to several administrative functions that you can use to integrate the QuantStudio 12K Flex Instrument into a laboratory workflow The following functions are available from the touchscreen and can be used after installation to customize the QuantStudio 12K Flex Instrument settings and configure it for network use Defining the date and time 0 eee eee eee eee eens 168 Defining the instrument settings e eee eens 168 Defining the maintenance reminders 0 00 0 cece eee eee 169 Defining the network settings 0 170 Defining the system shortcuts sua ssna sneer eee eens 171 Reviewing the instrument statistics 6 0 0 occ eee 171 Enabling disabling instrument security ee eee 172 Viewing the instrument log 0 e eee ee 173 Note The touchscreen does not provide access to all instrument functions Features such as instrument calibration and remote notification are available only through the QuantStudio 12K Flex Software Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 167 A Manual Instrument Operation Administering the instrument from the touchscreen Defining the date and time Defining the instrument settings 168 Applied Bios
353. tration User Guide 143 144 Security Audit and Electronic Signature Managing user accounts Permissions and default user roles The following table shows all user configurable permissions and the settings for the default user accounts Permissions Default user roles c 2 DEE Category Function amp E 2 v v E D 2 lt Setup Create and edit experiments or experiment Yes Yes Yes templates includes running experiments Run Perform a run using the Quickstart function Yes Yes Yes Start a run Yes Yes Yes Stop a run Yes Yes Yes Targets Create targets Yes Yes Yes Library Edit targets Yes Yes Yes Delete targets Yes No Yes Analysis Create analysis settings includes default settings Yes Yes Yes settings Edit analysis settings includes default settings Yes Yes Yes Library Delete analysis settings Yes No Yes Run Methods Create a run method Yes Yes Yes Library Delete a run method Yes No Yes Dye Library Create a custom dye Yes Yes Yes Delete a dye Yes No Yes Preferences Edit the system preferences Yes No Yes Export the system preferences No No Yes Import the system preferences No No Yes Calibrations Perform calibrations Yes Yes Yes RNase P Perform an RNase P experiment Yes No Yes Instrument Add or remove QuantStudio 12K Flex Instrument No No Yes Configuration from monitoring Security Configure the security and audit feature No No Yes
354. trument After each run the QuantStudio 12K Flex Software receives data in the form of a raw spectra signal for each reading It determines the contribution of each fluorescent dye used in the sample by comparing the raw spectra to the pure spectra calibration data When you save an experiment after analysis the QuantStudio 12K Flex Software stores the pure spectra with the collected fluorescence data for that experiment IMPORTANT Calibrate only those dyes that are present in the chemistries that you intend to run on your QuantStudio 12K Flex System Perform a dye calibration every year or as often as necessary depending on instrument use IMPORTANT Calibrate only dyes that are present in the chemistries that you intend to run on the QuantStudio 12K Flex System For example if you intend to runa TaqMan RNase P plate or array card to verify instrument performance see page 63 you must calibrate the FAM dye TAMRA dye and ROX dye because all three are present in the TaqMan assay chemistry IMPORTANT Perform a background calibration before every series of dye calibrations Because the age and use of instrument components can affect spectra readings we recommend performing a dye calibration at least every year System dyes The Applied Biosystems QuantStudio 12K Flex Real Time PCR System calibrates the following system dyes FAM dye NED dye ROX dye SYBR Green dye TAMRA dye a
355. ts Raw data Contains the raw data collected by the QuantStudio 12K page 213 Flex Instrument during the experiment run Amplification Contains the normalized data collected during the cycling page 214 data stage of PCR amplification which the QuantStudio 12K Flex Software uses to generate the amplification plot Note Not applicable for presence absence genotyping or melting curve experiments that are run without a PCR cycling stage Multicomponent Contains the spectral data used by the QuantStudio 12K page 215 data Flex Software to generate the multicomponent plot that displays the contribution of each dye over the duration of the PCR run Results data Contains the normalized processed and analyzed data page 215 generated by the QuantStudio 12K Flex Software Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 209 File header File Format Reference QuantStudio12KFlex export format The plate setup file begins with a header that describes the qualities of the QuantStudio 12K Flex Instrument used to run the experiment and several other general experiment properties Each line starts with an asterisk and ends with a carriage return in the following pattern lt field name gt lt field value gt Note The QuantStudio 12K Flex Software automatically removes any leading and trailing white space around the field name and field valu
356. ty control specifications are shipped An analysis setting for the Baseline Threshold algorithm You enter the baseline start and end cycles for the amplification plot See also baseline An analysis setting for the Baseline Threshold algorithm You enter the threshold value and select whether to use automatic baseline or manual baseline values The software uses the baseline and the threshold values to calculate the threshold cycle C7 A plot of data collected during the melt curve stage Peaks in the melt curve can indicate the melting temperature Tn of the target or they can identify nonspecific PCR amplification In the software you can view the melt curve as normalized reporter Rp vs temperature or as derivative reporter R vs temperature In a high resolution melting experiment you can view the melt curve as fluorescence vs temperature Also called dissociation curve The melt curve shape and the difference in melting temperature Tm values In the thermal profile a stage with a temperature increment to generate a melt curve The temperature at which 50 of the DNA is double stranded and 50 of the DNA is dissociated into single stranded DNA In a melt curve experiment the melt curve plot displays the melting temperature In Melt Curve experiments the region before and after the melting temperature Tm A plot of the complete spectral contribution of each dye for the selected well s over the duration of t
357. uantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 189 DI Command line Software Operation Overview Overview The QuantStudio 12K Flex Software includes a command line application that allows you to generate and export batches of experiment files from an MS DOS prompt or a batch file The application is intended for advanced users who choose to create or export experiments using a scripting language IMPORTANT After you use the command line application to generate experiment files validate the contents of the files by opening them in the QuantStudio 12K Flex Software Command line The command line interface supports the workflows in the following figure For each workflows workflow the figure shows both the required and optional supporting files Single Experiment File Creation Workflow QuantStudio 12K Flex Application Command line y Input gt Experiment Document SDS Setup Sample to Well AIF X File Barcode Template edt File txt File txt LU xml Export Workflow b Rrap QuantStudio 12K Flex Application Experiment Document Single eds Required 190 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Appendix D Command line Software Operation Supporting files for experiment creation Supporting files for experiment creation The file generation functi
358. udio 12K Flex Instrument to the network L ee 129 Required materials ass xa e a See OR EERE UY CL Wee ede ane 129 Defining the internet protocol settings cece eee eee eee 129 6 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Contents Connecting the computer to the network cece eee ence eens 130 Reg ired mMat ridlg x25 4 hie ae Settle rate eh oocee aa biden Wht ae a a 130 Computer requirement sssr SER a 0 0 X e whe dek ese dened Wee cee bd Sw Ree ee Bae es 130 Required Information cece cece eee eee eee e eee 130 Settingiupthe computers ccc2 notice ee e tet CHE eh da ie eh ahha t ea ends 130 Installing the QuantStudio 12K Flex Software 00 0 c cece eee eee eens 131 Monitoring a QuantStudio 12K Flex Instrument 0 000 cece cece een een eens 132 About remote monitoring eee eee eee eee eee e eens 132 Monitoring the status of an instrument during a FUN 200000 e cece cece eee 132 Uploading or downloading an experiment or template n n 0c eee eee 133 Enabling or changing the calibration reminders cece cece eee eee 134 S CHAPTER 6 Security Audit and Electronic Signature 137 Administrators Overview soassa sedanas uuaa eee eal bd ek beer det ee eae ee 138 Example applications r 0 c K ene ceeded R KT R 0 ed edd aed e dR A ee 138 Configuring the system security 2 2 ccc eee eee eee eee eee 139 Accessi
359. ument Twister Robott Automates loading and unloading of consumables to and from the QuantStudio 12K Flex Instrument Not for diagnostic use Connection Description Power cables Supply power to the computer the Applied Biosystems Twister Robot and the QuantStudio 12K Flex Instrument T LAN connection or Ethernet cable Connects the QuantStudio 12K Flex Instrument Ethernet port to the Ethernet port on the network interface card in the computer DVI cable Connects the monitor to the computer DVI port Barcode reader cable Connects the barcode reader to the computer USB port Keyboard cable Connects the keyboard to the computer USB port C D E F Mouse cable Connects the mouse to the computer USB port G Serial cable Connects the Twister Robot to the computer serial port Suppli Suppli es 115 230 V depending on the geographic location of the installation ed with the QuantStudio 12K Flex System Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 23 1 Getting Started QuantStudio 12K Flex System hardware QuantStudio 12K Flex System hardware Instrument The QuantStudio 12K Flex System consists of the components shown in the components following figures Front view Component Description A USB port
360. ument Operation Maintaining the instrument from the touchscreen Maintaining the instrument from the touchscreen The QuantStudio 12K Flex Instrument touchscreen provides access to several maintenance functions that cannot be accessed remotely from the QuantStudio 12K TM Flex Software The following local QuantStudio 12K Flex Instrument functions are performed as part of regular QuantStudio 12K Flex Instrument maintenance m Backing up and restoring the instrument settings 050 008 164 S Performing an instrument self test 165 S Updating the instrument firmware e e ees 166 Note The touchscreen does not provide access to all instrument functions Features such as instrument calibration and remote notification are available only through the QuantStudio 12K Flex Software Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 163 A Manual Instrument Operation Maintaining the instrument from the touchscreen Backing up and restoring the instrument settings You can use the QuantStudio 12K Flex Instrument touchscreen to back up the instrument settings icon standby time out and cover idle temperature and some network settings the Autodiscovery and Smart Monitoring options In the event that the QuantStudio 12K Flex Instrument settings are reset you can restore the settings from the backup The QuantStudio 12K Flex Instrument backs up
361. und each well area Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 43 Calibrating Multi Well Plate and Array Card Sample Blocks ROI calibration 8 After you inspect all ROI images verify the status of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or the data it collected is unusable Analysis status Action Passed Click Next then remove the plate or array card when the QuantStudio 12K Flex Instrument ejects the tray arm Failed Troubleshoot the failed calibration as described in Troubleshooting ROI calibrations on page 72 WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can reach 100 C Allow the plate to reach room temperature before removing IMPORTANT If the QuantStudio 12K Flex Instrument does not eject the plate remove the plate according to Troubleshooting ROI calibrations on page 72 9 Discard or store the plate or array card Consumable Action Array card Discard the array card if you do not plan to perform a uniformity calibration soon Note You can reuse the array card if the ROI and uniformity calibrations are performed on the same day Plate Return the ROI calibration plate to its packaging sleeve If you plan to perform background and uniformity calibrations e
362. ve 3 Vortex and centrifuge the plate a Vortex the ROI calibration plate for 5 seconds b Centrifuge the plate for 2 minutes at less than 1500 rpm IMPORTANT The ROI calibration plate must be well mixed and centrifuged c Confirm that the liquid in each well of the ROI calibration plate is at the bottom of the well If not centrifuge the plate again at a higher rpm and for a longer period of time Correct Incorrect Za Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time 50 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 2 Calibrating Multi Well Plate and Array Card Sample Blocks Uniformity calibration Performing the 1 In the Home screen of the QuantStudio 12K Flex Software click Instrument calibration Console 2 In the Instrument Console select your QuantStudio 12K Flex Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a QuantStudio 12K Flex Instrument to your list before you can manage it 3 After the QuantStudio 12K Flex Instrument is added to your list of instruments select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenance then click Uniformity b In the Uniformity Calibration screen click Start Calibration 5 Click Next then perf
363. ve adhered the plate to the heated cover within the instrument Power off the QuantStudio 12K Flex Instrument Wait for 15 minutes then power on the QuantStudio 12K Flex Instrument and eject the plate If the plate does not eject power off and unplug the QuantStudio 12K Flex Instrument then open the access door Wearing powder free gloves reach into the QuantStudio 12K Flex Instrument and remove the plate from the heated cover then close the access door Perform a background calibration to confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a local Life Technologies Field Service Office k Bunooysayqnos s4901g ajdwes pieg Aewy pue 3 eld N M MNW Bunesqneg 0IDHIG1UREnpH swas soig panddy wl aping Jas uonensiuIUpy pue aaueuajulew WajSkS Yd awl jay Xa4 YZ GL Table 5 Troubleshooting dye calibrations Problem symptom Possible cause Action One or more raw spectra are at or below the detectable threshold for the calibration Dye calibration plate was centrifuged insufficiently Dye calibration plate contains old or insufficient reagents If you are running a custom dye calibration plate the dye may not be present at a sufficient concentration 1 Unload the QuantStudio 12K Flex System and view the wells of the dye calibration plate If the liquid in the wells is not e
364. wer on the QuantStudio 12K Flex Instrument and eject the plate 3 If the plate does not eject power off and unplug the QuantStudio 12K Flex Instrument then open the access door 4 Wearing powder free gloves reach into the QuantStudio 12K Flex Instrument and remove the plate from the heated cover then close the access door 5 Perform a background calibration to confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a local Life Technologies Field Service Office sy20 g ajdues pueg Aewy pue ayeyg N M MNW Bunesqneg Z4aydeyg Bunooysayqnos Z 9L oipn s ueng swas soig panddy WL aping J SN uONesSiuiWpy DUS 32ULUIJUIEW W JS S YJd aw Jeay X2 4 YZ Table 6 Troubleshooting normalization calibrations Problem symptom Possible cause Action Normalization calibration failed Abnormally low spectra across all wells of the plate or array card 1 Confirm that you loaded an normalization plate or array card into the QuantStudio 12K Flex Instrument If not perform the calibration again using the correct normalization plate or array card 2 If you are using the correct plate or array card perform the calibration again using a different normalization plate or array card 3 If the calibration fails again contact Life Technologies technical support One or more wells produced spectra that deviate significantly f
365. wo T1 6A 250VAC 5 x 20 mm electrical fuses that protect the Twister Robot from excessive electrical current Rack parts and functions Racks are removable aluminum frames used as input and output locations for PCR consumables Rack positions are numbered counter clockwise with position 1 closest to the front of the Twister Robot see below Each rack is labeled for a specific position and cannot be exchanged with the other racks Component Description Handles For connecting or disconnecting racks from the pod B Rack locator Locks the rack onto the pod in the correct position notch Note Do not drop the racks If the rack is bent the Twister Robot cannot properly place the consumables 28 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Chapter 1 Getting Started 1 QuantStudio 12K Flex System hardware Electrical We recommend several devices to protect the QuantStudio 12K Flex System in protective devices environments with large voltage and power fluctuations Power line regulator We recommend the use of a 1 5 kVA power line regulator in areas where the supplied power fluctuates in excess of 10 of the normal voltage Power fluctuations can adversely affect the function of the QuantStudio 12K Flex System Note A power line regulator monitors the input current and adjusts the power supplied to the QuantStudio
366. x Instrument Ethernet port to the network interface card in the computer Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide 25 1 Getting Started QuantStudio 12K Flex System hardware Barcode reader The Applied Biosystems QuantStudio 12K Flex Real Time PCR System can include two barcode readers for data entry and plate recognition e A hand held barcode reader for scanning plates manually e A fixed position barcode reader for automatically scanning plates as they are loaded into the instrument available only with the Twister Robot Both barcode readers use 670 nm Class II lasers to scan plates and both readers are capable of reading Code 128 alphanumeric which supports 128 ASCII character barcodes The barcode readers are optional and available depending on the system configuration About the hand held barcode reader WARNING LASER HAZARD Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots Never look into the laser beam Remove jewelry and anything else that can reflect the beam into your eyes Protect others from exposure to the beam The optional hand held barcode reader functions as an extension of the keyboard You can use the reader to scan barcodes into the QuantStudio 12K Flex Software To scan a barcode using the hand held barcode reader 1 In the QuantStudio 12K Flex Software select the field where
367. y only one computer at a time A networked computer running the QuantStudio 12K Flex Software can transfer experiments to and from an instrument begin or stop a run and perform some maintenance functions During a run the Remote Monitoring feature of the software can be used to view the run status temperature and amplification data in real time See Monitoring a QuantStudio 12K Flex Instrument on page 132 for more information on remote monitoring Note Remote monitoring does not allow you to control the QuantStudio 12K Flex System The QuantStudio 12K Flex Instrument features a Gigabit Ethernet port for direct communication with the QuantStudio 12K Flex System computer and for network communication When the QuantStudio 12K Flex System is connected to a network computers on the network that run the QuantStudio 12K Flex Software can s Send and download experiments to and from the QuantStudio 12K Flex System s Run experiments on the QuantStudio 12K Flex System s Remote monitor the QuantStudio 12K Flex System as it performs runs The Ethernet port of the QuantStudio 12K Flex Instrument supports e Static IP network service with subnet mask primary and secondary data network service DNS and default gateway settings or dynamic host configuration protocol DHCP network service s mDNS DNS for local domains Note Because mDNS is limited to direct network connections a QuantStudio 12K Fl
368. you want to enter the barcode 2 Hold the barcode reader 20 30 cm away from a plate and aim at the center of the barcode then press the trigger Slowly move the scanning beam across the barcode until the reader emits a high pitched tone When the reader scans a barcode it automatically e Transmits the alphanumeric equivalent of the barcode to the QuantStudio 12K Flex Software The software enters the barcode text wherever the cursor is active e Transmits a carriage return character the equivalent of pressing the Enter key For more information on the hand held barcode reader see the barcode reader user documentation shipped with the QuantStudio 12K Flex System 26 Applied Biosystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide Twister Robot components Chapter 1 Getting Started 1 QuantStudio 12K Flex System hardware The QuantStudio 12K Flex System supports the use of the Applied Biosystems Twister Robot an optional QuantStudio 12K Flex System accessory that consists of the components shown below Note See the Applied Biosystems QuantStudio 12K Flex Real Time PCR System Automation Guide Part no 4470693 for information on operating calibrating maintaining and integrating the Twister Robot Front view Component Description Reach axis Moves the grip horizontally 28 5 50 1 cm 11 25 19 75 in from the center of the robot post Wrist
369. ypuanb Juadsaronyj uou ayy pue sadp Jay1odar LA PUL INVA 94 HLA paad ei sagoid ueber sypads ajayye Jo jas e Sursn PEEGTS4 S479812 JNS uO 101 s jdures suaaids zu ur dx ayy aise xa J NZL L OrPMISHUeND L uo UNL aq 01 yUauTtIadxa SutdAyouas e 101 paean apy das ayejd e smoys jdwex SurMo oj AY aOW OAN aOW OAN aOW OAN aOW OAN aOW OAN aOW OAN aOW OAN aOW OAN OW OAN aOW OAN aOW OAN aOW OAN squewog AqtTquend zAsyousend OIA WV OIA WYA DIA NY 3 OIA WWA OIA WWA OIA WWA 1911 0day RER 0 SZ 86 duu ddI 0 S7 86 9909 ddI 0 S7 86 9909 ddI 0 S7 86 duu 34T 0767789619909 gdi 0767789619909 DIN 0767789619909 DIN 0 S7 86 d9uu DIN 0 S7 86 d9uu DIN 0 S7 86 d9uu DIN 0 SZ 86 4984n DIN u 0 SZ 86 g98u SRL LOTO qobreLl s u w dx BuldAjoua9g OdI TTOO a dI TTOO a RER TTOO a RER FTOO a OdI FTOO a OdI FIOS a aueN qebszeL AOTOD dnozbotg wen dnozbotg ZoToD etdues u 0 0 SZ 999 TOST vL07 07671999 TOTQUOD vL07 07671999 TOTQUOD vL07 07671999 TOTQUOD vL07076Z1 999 TOTQUOD vL07076Z1 999 TOTQUOD u 0 0 GZ 9Yn TOTQUOD u 0 0 GZ dOd TOT UCD u 0 0 GZ G9 u TOTIUOD u 0 0 GZ 98u TOT UCD squemiog reyouend ZoTOD etTdues aure etdwes Tom dues ZTE ZTU ZTU TIU TIv TTIW ban ban ban AMTN OR ADEA T dnjes etdues XOU 9U I J eATSSed x XO9TANZTOTpnasquend sdAy quewnzqsult 0767 01999 ToT UCD
370. ystems QuantStudio 12K Flex Real Time PCR System Maintenance and Administration User Guide If the instrument is in standby touch the QuantStudio 12K Flex System touchscreen to activate it then press 4 Inthe Main Menu touch Settings then touch Set Date amp Time Inthe Set Date amp Time screen a Touch the Time zone field then touch the correct time zone from the list b Touch the Date field enter the current date then touch Done c Touch the Date Format drop down list then select the format for your region d Touch each Time field enter the appropriate time units then touch Done e Touch 12 Hour or 24 Hour to select the appropriate time format f Touch Save to save the settings then touch OK when prompted Touch a to return to the Main Menu Ifthe instrument is in standby touch the QuantStudio 12K Flex System touchscreen to activate it then press 4 In the Main Menu touch Settings then touch Configure the Instrument Touch the Instrument Name field enter up to a 16 character name for the QuantStudio 12K Flex Instrument then touch Done The instrument name is the alphanumeric string used to identify the QuantStudio 12K Flex Instrument on the network IMPORTANT To connect the QuantStudio 12K Flex Instrument to a network the name must be unique IMPORTANT The instrument name cannot include spaces or special characters such as lt gt 17 Upload

Applied Biosystems QuantStudio™ 12K Flex Real

Contents

Download Pdf Manuals

Related Search

Related Contents

Applied Biosystems QuantStudio&trade; 12K Flex Real

Contents

Download Pdf Manuals

Related Search

Related Contents

Applied Biosystems QuantStudio™ 12K Flex Real