DNF-915-K0500
Contents
1. Excess primer dimer species in sample Further dilute input DNA sample concentration with 1X TE buffer to minimize primer dimer interference and repeat experiment Sample peak s migrate after or co migrate with 5 000 bp Upper Marker DNA sample size out of range of assay Analyze samples with DNF 920 75 bp 15 000 bp or DNF 930 75 bp 20 000 bp reagent kit No sample peak or marker peak observed for individual sample Air trapped at the bottom of sample plate and or marker plate well or bubbles present in well Insufficient sample volume A minimum of 20 uL is required Capillary is plugged Check sample marker plate wells for trapped air bubbles Centrifuge plate Verify proper volume of solution was added to sample well Check waste plate for liquid in the capillary well If no liquid is observed follow the steps outlined in Appendix G Capillary Array Cleaning of the Fragment Analyzer User Manual for unclogging a capillary array Technical Support Technical Support and Contact Information 1 For questions with Fragment Analyzer operation or about the DNF 915 Reagent Kit contact AATI Technical Support by phone at 515 296 6600 or by email at support aati us com Rev DNF 915 2014MAR10 5 Advanced Analytical Technologies Inc Notes 6 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc2. 11 1 well DNA Ladder PUMBe nol Sanpies ech Run 96 Capillary 95 1 well DNA Ladder Total Electrophoresis Run Time 50 minutes 33 55 Array 80 minutes 55 80 Array DNA Sizing Range 35 bp 5 000 bp defined by lower upper marker 35 bp 100 bp lt 10 Separation Resolution 100 bp 2 000 bp lt 5 2 000 bp 5 000 bp lt 10 DNA Sizing Accuracy 5 or better DNA Sizing Precision 2 CV DNA Fragment Concentration Range 0 5 ng uL 50 ng uL input DNA adjustable by dilution of sample 1 Results using DNA Ladder or DNA Fragment standards initially prepared in 1X TE buffer Storage Conditions Store at 4 C Store at Room Temperature Store at 20 C DO NOT FREEZE DO NOT FREEZE FA dsDNA Gel Intercalating Dye Ca pulery Conditioning Solution oe ees 35 bp and 5 000 bp Markers Mineral Oil Buffer Dilution Buffer 1X TE 100bp Plus DNA Ladder Ensure all reagents are completely warmed to room temperature prior to use Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc Additional Material and Equipment Required Hardware Software and Reagents available from AATI 1 Hardware Fragnent Analyzer 12 capillary or 96 capillary CE system with LED fluorescence detection e 12 Capillary Array Cartridge Fluorescence 33 cm effective 55 cm total length 50 um ID part A2300 1250 3355 OR e 12 Capillary Array Cartridge Fluorescence 55 cm effective 8
3. It is highly suggested to perform one of the following methods to ensure complete mixing A When adding 2 UL of sample to the 22 UL of diluent swirl the pipette tip while pipetting up down to further mix B After adding 2 UL of sample to the 22 uL of diluent place a plate seal on the sample plate and vortex the sample plate at 3000 rpm for 2 min Any suitable benchtop plate vortexer can be used Ensure that there is no well to well transfer of samples when vortexing The plate should be spun via a centrifuge after vortexing to ensure there are no trapped air bubbles in the wells C After adding 2 UL of sample to the 22 UL of diluent use a separate pipette tip set to a larger 20 UL volume and pipette each well up down to further mix D Use an electronic pipettor capable of mixing a 10 UL volume in the tip after dispensing the 2 UL sample volume Some models enable using the pipette tip for both adding and mixing 4 After mixing the samples with 1X TE dilution buffer in each well check the wells of the sample plate to ensure there are no air bubbles trapped in the bottom of the wells If necessary centrifuge the plate to remove any air bubbles The presence of trapped air bubbles can lead to injection failures 5 Run the sample plate immediately once prepared or cover the sample plate with a cover film store at 4 C and use as soon as possible Alternatively to prevent evaporation place a mineral oil overlay on each sa
4. if the marker solution is loaded into a row other than Row A on a 12 capillary instrument this can be adjusted prior to or while the method is loaded on the experimental queue 6 To apply any adjustments to the method being placed on the experimental queue press the OK button To exit the editor screen without applying any changes press the Cancel button IMPORTANT Any edits made to the experimental method from the Separation Setup or Method Summary screen will only apply to the currently loaded experiment in the queue No changes are made to the original separation method file Processing Experimental Data 1 When processing data the PROS zxe 2 0 software Version 1 3 and higher will automatically recognize the separation method performed and apply the appropriate matching configuration file from the C PROSize 2 0 Configurations directory a The DNF 915 33 separation method will be processed using the DNF 915 33 DNA 35 5000bp configuration file b The DNF 915 55 separation method will be processed using the DNF 915 55 DNA 35 5000bp configuration file NOTE If the preloaded PROSize 2 0 software configuration files DNF 915 33 DNA 35 5000bp and DNF 915 55 DNA 35 5000bp are not located in the C PROSize 2 0 Configurations directory contact AATI Technical Support to obtain these files 2 The data is normalized to the lower marker set to 35 bp and upper marker set to 5 000 bp and calibra
5. Capillary instruments or larger volumes Corning 430776 available from Fisher 05 538 53 or VWR 21008 771 b 50 mL for 12 Capillary instruments or 50 mL volumes BD Falcon 352070 available from Fisher 14 432 22 or VWR 21008 940 Clean graduated cylinder for measurement of dsDNA Gel volume and dilution of 5X 930 dsDNA Inlet Buffer and 5X Capillary Conditioning Solution Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc Safety When working with chemicals always follow standard safety guidelines such as wearing a suitable lab coat disposable gloves and protective eyewear For more information about the specific reagents please refer to the appropriate material safety data sheets MSDSs that can be obtained from the product supplier Fragment Analyzer Start Up Instrument Preparation Gel Preparation 1 2 Store the dsDNA Separation Gel at 4 C upon arrival The Intercalating Dye is supplied as a 20 000X concentrate in DMSO and should be stored at 20 C NOTE For this assay it is recommended to use the Intercalating Dye at 2X normal concentration 1 10 000 dilution Bring the dsDNA Separation Gel and Intercalating Dye to room temperature prior to mixing Mix appropriate volumes of Intercalating Dye and Separation Gel necessary for less than two weeks of operation Use the supplied 50 mL conical centrifuge tube to allow a small minimum working volume For larger volumes use a 250 mL conica
6. mL Part FS SMO15 Dilution Buffer 1X TE 60 mL Part DNF 495 0060 Conical 250 mL Centrifuge Tube for prepared Separation Gel Intercalating Dye mixture DNF 915 dsDNA Reagent Kit 35 bp 5 000 bp 1000 Samples Part DNF 915 K1000 Kit Components 1 2 3 FA dsDNA Gel 35 bp 1 500 bp 500 mL Part DNF 810 0500 Intercalating Dye 30 uL x 2 Part DNF 600 U030 5X 930 dsDNA Inlet Buffer 300 mL dilute with sub micron filtered water prior to use Part DNF 355 0300 5X Capillary Conditioning Solution 100 mL dilute with sub micron filtered water prior to use Part DNF 475 0100 35 bp and 5 000 bp Markers 3 2 mL Part FS SMK480 0003 a 0 5 ng L concentration each in 1X TE buffer 100bp Plus DNA Ladder 1 0 mL Part FS SLR915 0001 a 100 bp 3 000 bp 2 5 ng uL total DNA concentration in 1X TE buffer Mineral Oil 15 mL Part FS SMO15 Dilution Buffer 1X TE 125 mL Part DNF 495 0125 Conical 250 mL Centrifuge Tube for prepared Separation Gel Intercalating Dye mixture Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc Application The DNF 915 Reagent Kit from AATI is for the analysis of dsDNA fragments between 35 bp and 5 000 bp Sizing and relative quantification between samples can be obtained using this kit Example applications include general PCR fragment sizing and QC and genotyping Specifications Sample Volume Required 2 uL adjustable depending upon sample concentration 12 Capillary
7. 0 cm total length 50 um ID part A2300 1250 5580 OR e 96 Capillary Array Cartridge Fluorescence 33 cm effective 55 cm total length 50 um ID part A2300 9650 3355 OR e 96 Capillary Array Cartridge Fluorescence 55 cm effective 80 cm total length 50 um ID part A2300 9650 5580 Software Fragment Analyzer instrument control software Version 1 0 2 or higher e PROSize 2 0 data analysis software Version 1 3 or higher Reagents e Capillary Storage Solution 100 mL AATI GP 440 0100 Equipment Reagents to Be Supplied by User 1 96 well PCR sample plates Please refer to Appendix C Fragment Analyzer Compatible Plates and Tubes in the Fragwent Analyze User Manual for a complete approved sample plate list Multichannel pipettor s and or liquid handling device capable of dispensing 1 100 uL volumes sample plates and 1000 UL volumes Inlet Buffer plate Pipette tips 96 well plate centrifuge for spinning down bubbles from sample plates Sub micron filtered DI water system for diluting the 5X 930 dsDNA Inlet Buffer and 5X Capillary Conditioning Solutions Fisherbrand 96 DeepWell 1mL Plate Natural Polypropylene part 12 566 120 Inlet Buffer and Waste plate Reagent Reservoir 50 mL VWR 82026 355 or similar for use in pipetting Inlet Buffer plates sample trays Conical centrifuge tubes for prepared dsDNA Gel Dye mixture and or 1X Capillary Conditioning Solution a 250 mL for 96
8. 1 or Gel 2 pump position Ensure the fluid line is positioned at the bottom of the conical tube to avoid introducing air bubbles which can cause pressurization errors 7 When adding Separation Gel to the instrument update the solution levels in the Fragment Analyzer instrument control software From the Main Menu select Utilities Solution Levels A menu will be displayed to enter in the updated fluid levels Figure 1 8 When switching applications e g between kits prime the appropriate gel fluid line after loading fresh gel dye mixture From the Main Menu of the Fragment Analyzer instrument control software select Utilities Prime Select the desired fluid line s Conditioning r Check Solution Volumes _ ull Record the solution volumes here Conditioning Solution Waste Check the fluid volumes before proceeding Ensure that the waste is empty and that the gel and conditioning solutions are full Volume mL Gel 1 50 05 Gel 2 4234 2 15 102 44 C o cma Solution name 910 NaOH Figure 1 Solution Levels menu Gel 1 or Gel 2 and press OK to purge the fluid line with fresh gel Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc r a Prime Fluid Selected Cycles 1 B Fill Rate 300 l s i E Gel2 Empty Rate 300 pL s Figure 2 Prime menu Inlet Buffer Preparation Store the 5
9. 12 of the sample plate Alternatively once the 100bp Plus DNA Ladder has been run under the experimental method and additional samples are to be run under the same experimental conditions the ladder can be imported in the PROS7ze 2 0 software enabling use of all 12 wells per row or all 96 wells of the sample plate Sample Plate Preparation 1 The protocol below assumes the sample is originally present in a typical PCR buffer matrix Depending upon the concentration of product or sample matrix it may be necessary to adjust the dilution and or adjust the injection voltage and time to avoid overloading of the DNA sample 2 Using a clean 96 well sample plate pipette 22 UL of 1X TE dilution buffer supplied with kit to each well in a row that is to contain sample If running the 100bp Plus DNA Ladder in parallel with the samples pipette 24 UL of the Ladder solution directly no dilution into the specified well of the sample plate or row to be analyzed see previous Section 3 Pipette 2 uL of each DNA sample into the 22 UL of 1X TE dilution buffer in the respective wells of the Sample Plate mix the contents of the well using the pipette by aspiration expulsion in the pipette tip Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc Important Sample Mixing Information When mixing sample with diluent solution it is important to mix the contents of the well thoroughly to achieve the most accurate quantification
10. A8 a9 Sampag Tray name Enter Tray Name Herel 10 A10 SampA10 u All SampAL 12 a12 affa pA12 Select Row CED Save Tray Save Selected Row Reset Row Resettray Run Selected Row F v Add to queue Edit method 53 Run Entire Tray Add to queue Edit method Capillary Array Conditioning After entering data Add to queue Edit method select Add to queue i OOK Gi OOUA XX 0 0 PSI LED Off G Vent Closed Waste Closed J Stage Buffer H TraylH Figure 3 Main Screen showing selection of sample row and entering sample information 3 After sample information for the row or plate has been entered under the Run Selected Row field press Add to queue The Separation Setup form will be displayed enabling the user to select the experimental method and enter additional information Figure 4 11 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc r a Separation Setup Method DNF 915 33 DNA 35 5000bp mthds Edt Gel Gei x Trayname Enter Tray Name here Folder Prefix Enter folder prefix here Copy results Notes Enter any notes here Figure 4 Separation Setup form to select experimental Method and enter tray folder information 4 Inthe Separation Setup pop up form left click the dropdown and select the appropriate preloaded experimental Method file The available methods are sorted by kit number
11. New Software Version Refer to red highlighted text for additional AA updates ADVANCED ai ANALYTICAL DNF 915 dsDNA Reagent Kit User Guide DNF 915 K0500 DNF 915 K1000 For use with the Fragment Analyzer Automated CE System Fragment Analyzer Software Version 1 0 2 PROSize 2 0 Software Version 1 3 Revised March 10 2014 NOTICE The DNF 915 kit number has replaced the DNF 910 15L80M kit number The kit components and performance characteristics have not changed Future orders should reference the new DNF 915 kit number Advanced Analytical Technologies Inc Ph 515 296 6600 2711 South Loop Drive Suite 4150 Fax 515 294 7141 Ames IA 50010 www aati us com Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc DNF 915 dsDNA Reagent Kit 35 bp 5 000 bp 500 Samples Part DNF 915 K0500 Kit Components 1 2 3 FA dsDNA Gel 35 bp 1 500 bp 240 mL Part DNF 810 0240 Intercalating Dye 30 uL Part DNF 600 U030 5X 930 dsDNA Inlet Buffer 125 mL dilute with sub micron filtered water prior to use Part DNF 355 0125 5X Capillary Conditioning Solution 50 mL dilute with sub micron filtered water prior to use Part DNF 475 0050 35 bp and 5 000 bp Markers 3 2 mL Part FS SMK480 0003 a 0 5 ng L concentration each in 1X TE buffer 100bp Plus DNA Ladder 1 0 mL Part FS SLR915 0001 a 100 bp 3 000 bp 2 5 ng uL total DNA concentration in 1X TE buffer Mineral Oil 15
12. Vent Closed Q Waste Closed C Stage Buffer H Tray1H Figure 5 Main Screen after selection of samples to the run queue 15 Once an experiment has been loaded onto the queue the user can view or edit the method Administrator level only can edit a method by pressing the Method Summary field To remove the method from the queue press the X button to view the stepwise details of the method press the double down arrow icon 16 The user may add a Pause or Prime step into the queue by right clicking the mouse while over the queue and selecting Insert Pause or Insert Prime 13 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc 17 The order of the experimental queue can be rearranged by dragging down individual entries Further information regarding the Method Queue operation is provided in the Fragment Analyzer User Manual 18 Once started the instrument will perform all the programmed experiments in the Method Queue uninterrupted unless a pause step is present Note that additional experiments can be programmed and added to the Method Queue at any time while the instrument is running if desired After completion of the last queued experiment the instrument stage will automatically move to the Store location 12 Capillary Systems Row H of the inlet buffer tray containing the Capillary Storage Solution 96 Capillary Systems Sample 3 location Viewing and Editing Experimental Methods 1 A User level o
13. X 930 dsDNA Inlet Buffer at 4 C upon arrival DO NOT FREEZE Bring the 5X 930 dsDNA Inlet Buffer to room temperature prior to mixing and use In a clean container add 20 mL of the 5X dsDNA Inlet Buffer per 80 mL of deionized sub micron filtered water Agitate to mix The entire bottle can be mixed to 1X concentration and stored at 4 C if desired Capillary Conditioning Solution Preparation 1 Store the 5X Capillary Conditioning Solution at room temperature upon arrival DO NOT FREEZE In a clean container e g 50 mL or 250 mL conical centrifuge tube add 20 mL of the 5X Capillary Conditioning Solution per 80 mL of deionized sub micron filtered water Agitate to mix The entire bottle can be mixed to 1X concentration and stored at room temperature if desired Once mixed place the 1X Capillary Conditioning Solution onto the instrument and insert the CONDITIONING fluid line Conditioning Solution pump position Ensure the fluid line is positioned at the bottom of the conical tube to avoid introducing air bubbles which can cause pressurization errors The 1X Capillary Conditioning Solution should be added to the system as use demands A typical 12 capillary experiment cycle consumes less than 4 mL a typical 96 capillary experiment consumes less than 35 mL When adding fresh 1X Capillary Conditioning Solution to the instrument update the solution levels in the Fragment Analyzer instrument control software From the Main Menu sel
14. and are linked to the directory containing methods for the currently installed capillary array length e g 33cm or 55cm Select the following method a Select DNF 915 33 DNA 35 5000bp mthds when the 33 cm effective 55 cm total short capillary array is installed b Select DNF 915 55 DNA 35 5000bp mthds when the 55 cm effective 80 cm total long capillary array is installed 5 Select the appropriate Gel line being used for the experiment Gel 1 or Gel 2 using the dropdown 6 The Tray Name can be entered to identify the sample plate The Folder Prefix if entered will amend the folder name normally a time stamp of HH MM SS from the start of the CE run 7 To copy the experimental results to another directory location in addition to the default save directory C AATI Data check the Copy results box and select the desired Copy path directory by clicking the button and navigating the desired save directory 8 Any Notes can be entered regarding the experiment they will be saved and displayed in the final PDF report generated by the PROS7ze 2 0 software 9 Once all information has been entered press OK to add the method to the instrument queue press Cancel to abort adding the method 10 Repeat Steps 3 9 for any remaining sample rows to be analyzed 12 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc 11 On 96 capillary systems or in 12 capillary systems if the entire 96 well sampl
15. e tray is to be run using the same experimental method under the Run Entire Tray field press Add to queue A form similar to Figure 4 will be displayed for entering information and adding the run to the instrument queue for the entire 96 well sample tray 12 After a row or tray has been added to the queue the method s will be listed on the main screen under the Method Queue field Figure 5 13 Prior to starting the experiment verify all trays buffer storage waste marker sample etc have been loaded into their respective drawer locations pana 14 Press the Play icon amp to start the sequence loaded into the queue To Pause the queue after the currently running experiment is completed press the Ut button To Clear the run a _ A Fragment Analyzer 2555 User ID aati Database SysLog sdf ol a ia File Admin Utilities Help Sample ID SampD1 sampD2 Sampb3 SampD4 Sampos paya To start running SampD7 OOOOO0000000 Sampbe the queue press 1712345678 930 1 f2 SampD9 Tayname Enter Tray Name Here sampD10 the Play button SampD11 SampD12 A B D E F G H Load from File Save Trey Save Selected Row ResetRow Reset tray Run Selected Row Add to queue Edit method Run Entire Tray Add to queue Edt method Capillary Array Conditioning Add to queue Edit method Sy OOkV SF 00uA 0 0 PSI LED Off Q
16. ect Utilities Solution Levels A menu will be displayed to enter in the updated fluid levels Figure 1 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc Instrument Preparation 1 Check the fluid level of the waste bottle and waste tray daily and empty as needed 2 Prepare a fresh 96 DeepWell 1mL Plate filled with 1 0 mL well of 1X 930 dsDNA Inlet Buffer daily 12 Capillary System Row A only 96 Capillary System All Rows Do NOT overfill the wells of the inlet buffer plate 3 12 Capillary Systems In Row H of the same prepared buffer plate place 1 1 mL well of Capillary Storage Solution AATI GP 440 0100 Row H of the buffer plate is used for the Store location and the array moves to this position at the end of the experimental sequence 4 96 Capillary Systems In the Sample 3 drawer place a sample plate filled with 100 UL well of Capillary Storage Solution AATI GP 440 0100 Sample 3 is used for the Store location and the array moves to this position at the end of the experimental sequence IMPORTANT Ensure Row H of the buffer tray 12 capillary systems or Sample 3 96 capillary systems is always filled with Capillary Storage Solution and the capillary array is placed against Storage Solution when not in use to prevent the capillary tips from drying out and potentially plugging 5 Place the prepared inlet buffer plate into Drawer B top drawer of the Fragment Analyzer Ensure
17. in 1X TE buffer matrix to maximize sizing accuracy Whenever making adjustments to the sample dilution ensure the Dilution Factor of the PROS7ze 2 0 software is adjusted accordingly when processing the data Performing Experiments Running an Experiment 1 To set up an experiment from the Main Menu of the Fragment Analyzer instrument control software select the Operation tab Figure 3 Select the sample tray location to be analyzed 1 2 or 3 by left clicking the Sample Tray dropdown or by clicking the appropriate sample plate tab alternate plate view and choosing the appropriate location 96 Capillary Systems Note that Sample 3 is typically assigned to the Capillary Storage Solution 2 Left click a well of the desired sample plate row with the mouse The selected row will be highlighted in the plate map e g Row A in Figure 3 Enter the sample name if desired into the respective Sample ID cell by left clicking the cell and typing in the name Alternatively sample information can be imported from txt or csv file by selecting the Load from File option A Fragment Analyzer 2555 User ID aati Database SysLog sdf eae Soe i lt a TEZJE AA Eile Admin Utilities Help loaa Lege Operation Run Status abi Suu ate A B Manually enter Sample ID data OR load E from file option of save information by i an sampar Save Tray or Save Selected Row as Samp
18. ion Voltage 500 kV Time 10S Sec Vacuum Injection Pressure 20 PSI E Rinse Tray Buffer Row A Dips 1 7 Sample Injection Voltage Injection Voltage 5 00 kv Time 10 2 Sec 5 Vacuum Injection Pressure 20 PSI V Separation Voltage 60 kV Time 50 0 Min ok Cancel Figure 6 DNF 915 33 dsDNA Reagent Kit 35bp 5000 bp method 4 Figure 7 shows the preloaded method for the 55 cm effective 80 cm total length long array The Prerun and Separation voltage is set to 9 kV the Injection voltage to 7 5 kV and the Separation time to 80 min E Separation Method Gel Selection Gel 1 X V Perform Prerun Voltage 90S kV Time 30 54 Sec E Rinse Tray Buffer Row A Dips 15 7 Marker Injection Row A x Voltage Injection Voltage 7 50 kV Time 10S Sec Vacuum Injection Pressure 20 PSI E Rinse Tray Buffer Row A Dips 1 V Sample Injection Voltage Injection Voltage 7 50 kV Time 10 Sec D Vacuum Injection Pressure 20 PSI 7 Separation Voltage 90 kV Time 80 0 i gt Min ok cca Figure 7 DNF 915 55 dsDNA Reagent Kit 35bp 5000 bp method 1 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc 5 An Administrator level user has the option to adjust the Gel Selection Prerun settings Rinse settings including Tray Row and Dips Marker Injection settings including Row Sample Injection settings and the Separation settings For example
19. l centrifuge tube and remove the collar of the tube holder in the instrument reagent compartment For maximum accuracy it is recommended to dispense Separation Gel into a clean glass graduated cylinder for volume measurement and transfer to the working tube prior to adding Intercalating Dye NOTE Some loss of detection sensitivity will be observed over a two week period after the gel dye mixture has been prepared For best results it is recommended to prepare gel dye mixture daily It is not recommended to use gel dye mixture that is more than two weeks old The volume of Separation Gel required per run varies between 12 capillary and 96 capillary Fragment Analyzer systems The volumes required are summarized below For 12 capillary Fragment Analyzer systems ak E Volume of Intercalating dye Volume of Separation Gel analyzed 12 1 0 uL 10 mL 24 EA 15 mL 36 20 ul 20 mL 48 2 5 uL 25 mL 96 4 5 uL 45 mL A 5 mL minimum volume should be initially added to the tube For 96 capillary Fragment Analyzer systems Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc n a Volume of Intercalating dye Volume of Separation Gel analyzed 96 4 0 pL 40 mL 192 8 0 uL 80 mL 288 12 0 pL 120 mL 384 16 0 pL 160 mL 480 20 0 pL 200 mL 6 Place the prepared Separation Gel Intercalating Dye mixture onto the instrument and insert into the desired gel fluid line Gel
20. mple 20 UL well 6 To run the samples place the plate in one of the three sample plate trays Drawers 4 6 from the top of the Fragment Analyzer instrument Load or edit the experimental method as described in the following sections 7 The CCD detection system of the Fragment Analyzer system provides a high dynamic range for detection An ideal injection range would yield peak heights from 100 20 000 RFUs Overloading of sample can decrease separation resolution and saturate the detector leading to mismatched lower upper marker peak heights and poor results It is important to optimize sample dilution and concentration and use experimental parameters to work with within the specified RFU range 8 TIP If the above method 2 UL sample 22 uL 1X TE diluent yields peak heights consistently above 20 000 RFUs decrease the marker sample injection time or reduce the sample volume to 1 UL sample 23 UL 1X TE If low signals are encountered increase the marker sample injection time or alternatively add 4 UL of sample 20 uL of DI water in each well When making adjustments to the sample dilution the total volume should be maintained to at least 24 uL and whenever possible the chloride Cl salt content of the final sample should be adjusted to 10 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc approximately 10 mM CI to best match the 100bp Plus DNA Ladder and 35 bp 5 000 bp marker solution both are
21. nced Analytical Technologies Inc 3 The Marker solution is supplied as a ready to use solution containing 0 5 ng uL of each fragment in a 1X TE buffer solution It is intended for use as an external standard marker plate 4 Prepare the Marker solution plate by dispensing 30 UL well into Row A only 12 Capillary or every well 96 Capillary of a separate sample plate Cover the wells with 20 UL well of the supplied mineral oil to allow reuse for at least 30 injections 5 The prepared Marker solution plate should be placed into Drawer M third from top of the Fragment Analyzer Ensure that the plate is loaded with well A1 toward the back left on the tray 100bp Plus DNA Ladder Preparation 1 Store the 100bp Plus DNA Ladder solution at 20 C upon arrival 2 Bring the 100bp Plus DNA Ladder solution to room temperature prior to use agitate solution to ensure it is properly mixed and centrifuge vial prior to dispensing 3 The 100bp Plus DNA Ladder solution is supplied as a ready to use solution containing approximately 2 5 ng WL total DNA concentration in a 1X TE buffer solution It is used for calibrating the size of analyzed DNA fragments and is typically added to a well of the sample plate and run in parallel with the samples 12 Capillary Systems Pipette 24 uL of 100bp Plus DNA Ladder solution into well 12 of each row of the sample plate 96 Capillary Systems Pipette 24 uL of 100bp Plus DNA Ladder solution into well H
22. perator can View the steps of the experimental method by pressing the View link on the Separation Setup screen or by pressing the Method Summary option once a method has been loaded onto the experimental queue User level operators cannot edit any steps of a queued separation method 2 Administrator level operators can Edit certain steps of the experimental method To open the method editor screen press the Edit link from the Separation Setup screen Figure 4 The method editor screen is displayed showing the steps of the method Figure 6 3 The preloaded optimized steps for the DNF 915 33 Figure 6 and DNF 915 55 Figure 7 methods are shown below The general steps of the method are as follows 1 Full Condition flushing method Automatically enabled Gel Selection Gel 1 2 Perform Prerun ENABLED 6 kV 30 sec 3 Rinse DISABLED 4 Marker Injection ENABLED Voltage Injection 5 kV 10 sec This step injects the 35 bp 5 000 bp marker plate Rinse DISABLED 6 Sample Injection ENABLED Voltage Injection 5 kV 10 sec This step injects the prepared sample plate 7 Separation ENABLED Voltage 6 kV 50 min This step performs the CE Separation ol 14 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc r Separation Method Gel Selection Gel 1 X V Perform Prerun Voltage 60 kV Time 30 44 Sec E Rinse Tray Buffer Row Dips 1 7 Marker Injection Row A X Voltage Inject
23. r and 5 000 bp upper marker A total of 16 peaks should be observed with the sizes annotated as in Figure 8 All fragments in the ladder should be resolved 35248 NF 915 Kit 100 bp Plus DNA Ladder z So 15 30000 S oe 35 bp LM 5000 bp UM 20000 c LM 15000 E m t Q o 92o o g a 3 R SSISSS S yy 10000 a ALA AO ANAAL I 395 7 7 T T 7 i T 1 00 05 00 00 10 00 00 15 00 00 20 00 00 25 00 00 30 00 00 35 00 00 40 00 00 45 00 00 50 00 Time HH MM SS Figure 8 Representative 100 bp Plus DNA Ladder result injected with 35 bp lower marker and 5 000 bp upper marker using the Fragment Analyzer system with the DNF 915 reagent kit Method DNF 915 33 short array Figure 9 shows the same separation performed on a 55 cm effective 80 cm total length capillary array 3 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc 15472 14000 DNF 915 Kit 100 bp Plus DNA Ladder 500 12000 a a LM 100 200 300 400 4000 L 240 oo o o oo o o o Ss Ssshaans S o Raaj rS UM 1000 5000 bp UM WDE 1 1 1 1 I I I 1 1 1 00 20 00 00 25 00 00 30 00 00 35 00 00 40 00 00 45 00 00 50 00 00 55 00 01 00 00 01 05 00 01 10 00 01 15 00 01 20 00 Time HH MM SS Figure 9 Representative 100 bp Plus DNA Ladder result injected with 35 bp lower marker and 5 000 bp upper marker using the Fragment Analyzer system with the DNF 915 reagent kit Method DNF 915 55 long a
24. rray Troubleshooting The following table lists several potential assay specific issues which may be encountered when using the DNF 915 Reagent Kit and suggested remedies For a full list of instrument specific troubleshooting information refer to the Troubleshooting and Maintenance Guide for the Fragment Analyzer system Issue Cause Corrective Action A The peak signal is gt gt 1 Input DNA sample 20 000 RFU upper concentration is too marker peak is low or high not detected relative to lower marker Further dilute input DNA sample concentration with 1X TE buffer and repeat experiment 2 Reduce injection time and or injection voltage and repeat experiment Use the same injection voltage time settings for the Marker Plate and Sample Plate to maximize quantification accuracy Rev DNF 915 2014MAR10 4 Advanced Analytical Technologies Inc B No peak observed for DNA sample when expected Lower Upper Marker peaks observed Sample concentration too low and out of range Sample was not added to 1X TE diluent or not mixed well Prepare more concentrated sample and repeat experiment e g 4 uL sample 20 uL DI water OR Repeat experiment using increased injection time and or injection voltage for Marker Plate and Sample Plate Verify sample was correctly added and mixed to sample well Sample peak s migrate before or co migrate with 35 bp Lower Marker
25. ted to the 100bp Plus DNA Ladder run in parallel to the samples Figure 8 shows an example of the 35 bp and 5 000 bp markers injected with the 100bp Plus DNA Ladder A total of 16 peaks should be observed 3 When processing data the PROS7ze 2 0 software is set to the DNA mode in the Advanced Settings The Quantification settings are set to Use Upper Marker for quantification with a Conc ng uL of 0 5 and a Dilution Factor of 12 2 UL sample 22 UL Diluent Marker Note that if a pre dilution was performed prior to the experiment the Dilution Factor setting should be changed to reflect the estimated final sample concentration 4 For full information on processing data refer to the PROS7ze 2 0 User Manual 2 Rev DNF 915 2014MAR10 Advanced Analytical Technologies Inc Fragment Analyzer Shut Down Storage Instrument Shut Down Storage The instrument automatically places the capillary array in the Store position against Capillary Storage Solution 12 Capillary Systems Row H of the buffer tray 96 Capillary Systems Sample 3 after each experiment no further action is required If the instrument is to be idle for more than one day turn off power to the system to preserve lamp lifetime Typical Separation Results 100 bp Plus DNA Ladder 1 Figure 8 shows the typical expected results for the 100 bp Plus DNA Ladder provided at an input total DNA concentration of 2 5 ng UWL in 1X TE buffer co injected with the 35 bp lower marke
26. that the plate is loaded with well A1 toward the back left on the tray 6 Place an empty 96 DeepWell 1mL Plate into Drawer W second from top of the Fragment Analyzer This plate serves as the capillary waste tray and should be emptied daily Alternatively the supplied open reservoir waste plate may be used Marker Ladder Sample Preparation General Information 1 The recommended 96 well sample plate for use with the Fragment Analyzer system is a semi skirted PCR plate from Eppendorf 951020303 Please refer to Appendix C Fragment Analyzer Compatible Plates and Tubes in the Fragment Analyzer User Manual for a complete approved sample plate list The system has been designed to operate using these dimensions styles of PCR plates Plates with similar dimensions may be used but note that capillary damage may occur with the use of poor quality PCR plates IMPORTANT Contact AATI if a different vendor or style of PCR plate is to be used in order to verify compatibility The use of PCR plates with different dimensions to the above recommended plate could possibly damage the tips of the capillary array cartridge 35 bp 5 000 bp Marker Preparation 1 Store the 35 bp and 5 000 bp Marker solution at 20 C upon arrival 2 Bring the 35 bp and 5 000 bp Marker solution to room temperature prior to use agitate solution to ensure it is properly mixed and centrifuge vial prior to dispensing Rev DNF 915 2014MAR10 Adva
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