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Pippin Prep Operations Manual

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1. side of cassette DN buffer volumes are Low AA side of cassette Figure 6 1 Low buffer levels in cassette buffer chambers 3 Inspect the gel columns Look for obvious breakage of the agarose column in each channel Important f there is obvious breakage do not use the lane Remaining lanes can be used 6 1 888 744 2244 6 www sagescience com e support sagescience com 4 Inspect bottom of cassette for bubbles in the detection region of the gel columns If a cassette has been jarred during shipping the agarose column can delaminate from the plastic bottom of the separation channel forming a thin flat bubble between the gel column and the bottom of the channel If this happens in the region used by the optical detector see illustration DNA detection will be extremely unreliable If a lane with such a bubble ts used for the reference marker the markers will not be properly identified and sample collection will occur at the wrong time or fail altogether Only the optical properties of the channel are affected electrophoretic properties of the channel are unaffected since the DNA travels through the center of the gel column above the bubble To find such bubbles turn the cassette upside down and view the bottom of the channels under a strong light while tilting the cassette back and forth see Figure 5 2 below Figure 5 3 shows the region of optical detection capture mode applications It may be used for
2. Control DNA for 0 75 Band Capture Ext Mkr 3 kb fragment 5 ug load 4 loads CBC7501 Reagent Replacement Kits Ethidium Bromide with External Marker Catalogue No Reagent Replacement Kits Dye Free with Internal Standards Catalogue No CDF3010 reagent replacement kit for 10 cassettes Marker H CDF2010 reagent replacement kit for 10 cassettes Marker L SDF2010 reagent replacement kit for 10 cassettes Marker G CDF1510 reagent replacement kit for 10 cassettes Marker K Reagent Replacement Kits Dye Free with External Marker Catalogue No CEF2010 reagent replacement kit for 10 cassettes Marker E 19 2 888 744 2244 6 www sagescience com 6 support sagescience com 20 Instrument Specifications Specifications for Pippin Prep Instrument Electrophoresis Voltage 100 V or 150V constant Typical current per sample lane 2 0 mA Optical detection 535 nm excitation 640 nm emission Power Requirements 100 240 VAC 10 VAC 2 5 A 50 60 Hz Weight 15 lbs 7 kg Dimensions 7H X 11W X 21D in 18H X 28W X 53D cm Approvals CE CSA Country of Origin United States 20 1 888 744 2244 6 www sagescience com 6 support sagescience com 21 Specifications for Gel Cassettes Dye Free Cassette Specifications Minimum Sample Load low single nanograms Maximum Sample Load 2 ug sheared genomic DNA Reproducibility specified on following pages Sample Recovery 50 80 1 Upto 10 ug of DNA may be loaded however a
3. Note There is a delay between the time that a fragment is detected and visible on the graph or 14 2 888 744 2244 6 www sagescience com 6 support sagescience com 19 Protocol Name Protocol Parameters Optical Signal Analyzing Runs Log Review Tab The default screen on the Pippin Prep is a tabbed format The Log Review is the third tab Overview The Log review screen is used to review data and programming parameters from previous size selection runs All programming parameters are displayed as originally entered in the Protocol Editor Optical and electrical current data from the run are displayed in graphical windows Specific regions of either window can be selected and expanded as needed for detailed analyses These data are useful for refining size selection performance and verifying instrument performance Log Review yo T Log file Directory Cassette Type DNA Marker Times and Values Current Trace D IA ra nN Dran O naratinne N N anual sco fhuara v A 12 O B O l Ip U IN Fre U NA p erations ivianual software V o lo LDI 9 888 744 2244 6 www sagescience com 6 support sagescience com Opening a Log file in the Review Screen Note A stand alone Review Log Program Is available for PCs Contact sage science support 1 Click the Log Review tab along top of the screen 2 To select a log file press the folder icon in next to the Log File field Log File ee l 3 Se
4. 1 1 1 1 1 I 1 1 1 1 1 00 00 00 45 00 30 00 45 01 00 0115 01 30 01 45 0200 0215 0230 Tie Bhan CALIBRATE SNAPSHOT INFO SHUTDOWN sage science 1 If switching from a ethidium bromide cassette to a dye free cassette rinse the electrode array by filling the rinse cassette with water placing the rinse cassette onot the optical nest and closing the lid for 20 seconds 2 Press CALIBRATE on the Control Panel TEST START CALIBRAT SMAP SHOT IMFO SHUTDOWN p 5 1 888 744 2244 6 www sagescience com e support sagescience com An LED Calibration window will launch The Calibration Status field will contain the message Calibration not done Place the Calibration fixture onto the optical nest so that all five LED detectors are covered The dark side of the fixture must be down closest to the LEDs Top Bottom Correct Position on Optical Nest Pippin Prep Operations Manual software v 6 13 CD9 5 2 888 744 2244 6 www sagescience com e support sagescience com 5 Close the lid 6 Check that the LED target setting is 0 80 Make sure 0 80 is entered in this field LED Calibration Target l ph mA Iteration LED Current counts 0445 1091 1949 0819 0606 Photocurrent mA oso g oso g oeo go oeo Calibration Status Calibration OK Prompt To restart calibration CALIBRATE To exit calibration window EXIT cec MN 7 Press CALIBRATE 5 3 888 744 2244 6 w
5. ethidium bromide 100 600 bp Marker B Product Number CSD2010 Use Cassette Definition 2 Agarose Marker B DNA Marker B Typical times to detector 20 75 150 I I I I I l I 00 40 00 50 01 00 01 10 01 20 01 30 01 40 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets Time to Time to Collect bp min 150 0 7 00 Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Specifications subject to change without notice see page 21 1 for specification definitions 21 3 888 744 2244 P www sagescience com e support sagescience com 21 3 2 Agarose ethidium bromide 25 ul elution well 50 600 bp Marker SSQ for EpiCentre ScriptSeq v2 protocol Product Number SQ2010 Use Cassette Definition ScriptSeq V2 DNA Marker SSQ Typical times to detector l l l l l 00 30 00 45 01 00 01 15 01 30 01 45 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets ScriptSeq Step Time to Time to Collect bp min ila il abn a Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Specifications subject to change without notice see page 21 1 for specification definitions 888 744 2244
6. 4 sage science Pippin Prep DNA Size Selection System Operations Manual Software v 6 13 Cassette Definition Set 9 888 744 2244 6 www sagescience com support sagescience com Sage Science Inc Suite 2400 500 Cummings Center Beverly MA 01915 2014 Sage Science Inc All rights reserved Sage Science and Pippin Prep are trademarks of Sage Science Inc All other brands and name mentioned herein are property of their owners 888 744 2244 6 www sagescience com e support sagescience com oof OND 6 2 6 3 9 1 9 2 9 3 9 4 10 10 1 10 2 10 3 10 4 10 5 11 11 1 11 2 11 3 11 4 TABLE OF CONTENTS INTRODUCTION sedensdategesetatedebadnsesebeunocaiedsteunlcuusdansecasecatedatecstadanadetaducustacuienntounies 1 1 SAFETY AND PREG AUTIONS sss s sss sss es essen e nenen enen 2 1 UNPACKING AND INSTALLATION ss sss s sss sss sss sss ssseseeeee nenen 3 2 ACHIEVING BEST RESULTS FROM THE PIPPIN PREP ss sss sss sss ss sss ss s lt 4 1 OPTICAL CALUIBRATION s ssss sss sss sss esse eenn ennenen eena nenen ee 5 1 PREPARING WB atc ce ice teeta ence eee eed dee ence ee emai eee 6 1 Visually Inspect the Cassette giicccecctccccses ence ctcsecieceas enacencetesctssreusectenstvedesveuccensssvessveneccteteneseetesneustic 6 1 Prepare the Cassette for Loading ssssssssssssssssssssseessssssseeeeeeeneee ennenen eenn nne 6 3 OREN TEST H 6 4 SAMPLE ra er BIN ert ease cet a eg dea eena 7 1 LOADING SANMDPLEBS sssss
7. Flash Drive prior to removal Failure to do so may cause data loss and problems with the upgrade procedure 8 Press EXIT to return to Main Menu This will install the software Installation may take up to 30 seconds EalT 9 To check that the software instrument has been update Press INFO again In the Information window the software version number is listed on the left side Instrument Name Pipl Sy Version 5 30 IPF Address 12 O Note The system should be ready to use re boot is not necessary Pippin Prep Operations Manual software v 6 13 CD9 17 4 888 744 2244 6 www sagescience com 6 support sagescience com 18 Warranty and Service Warranty Sage Science will offer a no charge repair or replacement of defective product if notified within the warranty period and product has not been misused altered or damaged by disaster This warranty is not transferable from the original purchaser to a subsequent purchaser Instruments The Pippin Prep instrument and monitor have a standard 2 year parts and labor warranty Instruments are subject to depot repair or replacement On site maintenance or repair is not implied Do not attempt to repair or disassemble the Pippin Prep instrument This will void the remaining warranty and possible create a dangerous condition Sage Science support staff may ask for a log file or image file to evaluate or troubleshoot instrument issues High Voltage The Pippin Pre
8. Relatively low input sheared DNA sample with broad size distributions 300 500 bp in lanes 2 5 Sample loading is the same in all lanes but different DNA ranges were eluted Brighter bands are ethidium bromide fronts due to elution 4 3 888 744 2244 6 www sagescience com e support sagescience com 5 Optical Calibration The Pippin Prep optical system should be calibrated for the specific type of cassette to be used dye free and ethidium bromide containing The instrument is supplied with a calibration fixture that fits into the optical nest If a lab is using only one type of cassette the optics should be calibrated daily when the instrument is in use If users switch cassette type that is to be run e g run a dye free cassette and then a ethidium bromide cassette or vice versa the optics should always be re calibrated prior to running the new type Main Tab Protocol Editor Log Review File Manager l System Options Lane Status Sample ID Current Elution Timer Reference Idle Separate Elute Display 4 0 00 J 00 00 00 1 P d 3 0 00 J 00 00 00 1 P 4 4 1 0 00 J 00 00 00 1 E 4 i Protocol 00 00 00 15 00 30 00 45 01 00 01 15 01 30 01 45 02 00 02 15 02 30 Time hh mm St at u S Protocol Name P l m ag 5 protocol xyz S an S Progress hh mm ss Display Voltage Y Clock 99 9 07 Feb 2012 17 07 31 Remote Access Estimated Completion Time 07 Feb 2012 18 06 49 Control Panel
9. Validation Control DNA can be purchased from Sage Science to check the performance of the Pippin Prep system The Control DNA is useful to test refine and troubleshoot Pippin Prep size fractionation protocols For comparison to factory tested cassettes an extraction protocol a typical Agilent Bioanalyzer result is provided with the kits Figure 12 1 shows the DNA profile and extraction protocol for the 2 agarose control kit and Figure 12 2 shows a typical result from a 150 bp tight extraction from the DNA control Kit Pippin Prep cassettes and instruments are functionally tested using restriction digests of genomic DNA from E coli For each cassette type a different restriction digest is used chosen so that size distribution of the digested DNA closely matches the useful fractionation range of the cassette without any significant peaks or discontinuities Following restriction digestion the control DNA is purified by phenol chloroform extraction dialyzed and diluted into Pippin Prep electrophoresis buffer without Ethidium bromide The DNA is premixed with Pippin Prep loading solution and is provided ready for loading no additional loading solution should be added The DNA concentration is 5 micrograms per 40 microliters 40 microliters of control DNA should be used per lane Each tube contains sufficient volume for 16 sample loads Marker bp 20 75 150 300 600 00 00 0045 00 30 00 45 01 00 0115 01 30 01 45 0200 02145 02 30 Tim
10. cassette definition file and press SELECT See Appendix A for summary of marker types and calibration 0 79 Marker D Target fight cuts only 1 3 OF Marker J 1 5 Marker A No Overflow Detection ev Marker B No Overflow Detection ZEE Marker E ve a Marker Band Capture only ir Marker C No OY erma Detection 3 DF Marker H EXPAND ALL COLLAPSE ALL CANCEL O Note No Overflow Detection is a legacy designation This refers to cassettes that are run with open elution modules large collections can cause modules fill and possibly overflow if not sealed with tape Software limits previously prevented mis programming and the No Overflow definitions were provided for user who used tape to seal the modules Module sealing tape is now provided with each cassette package and is recommended for all protocols No Overflow Detection is effectively included with each other existing cassette type and future cassette type but the term will no longer be used 888 744 2244 6 www sagescience com 6 support sagescience com 11 4 Selecting a Run Time Protocols can be run with widely variable run times up to 7 hours for low voltage runs The Run Time Selector consists of a field and a check box with the following default settings Run Time hh mm End Run when Elution is Completed B The 8 hour default value in the Run Time field ensures that a run is not unintentionally ended early The checked box in the End Run
11. collections Select the Peak programming mode Click the Peak button for the lane from which the fragment peak will be extracted Enter a value in for the BP Threshold Enter a base pair value that precedes the beginning of peak band of the fragment to be collected If possible use a threshold value that is lt 90 of the beginning of the leading edge of the target band The Pippin Prep will automatically collect the next peak that is detected The mass of minimum detectable band is approximately 50 ng Peak collection cannot be used with dye free cassettes Important t is important to verify the size of the band by electrophoresis Agilent Bioanalyzer agarose gel Also higher inout mass will cause bands to migrate faster due to a lower dye DNA ratio For very high loads gt 0 5ug band choose lower threshold values 11 11 888 744 2244 6 www sagescience com e support sagescience com 4 Enter Sample ID information Tight Range Time Peak Ref Lane BP Target BFP Stat BP End BP Pause T Start T End TPause BP Thresh off Sample ID Template off ff 600 Sample 1 5 Save After programming a new protocol or editing an existing one the protocol file must be saved prior to use If the protocol is new press Save If a protocol has been edited a yellow alert will be displayed in the Protocol Changes Not Applied field Save will save the file under the previously saved name and Save As
12. com 7 Ifthe optics have already been calibrated press START LED Calibration Target ph mA Iteration LED Current counts Photocurrent mA 0 09 Calibration Status Prompt Load TARGET cassette close lid and CALIBRATE To start without calibration load TARGET cassette and START To cancel calibration and protocol CANCEL CALIBRATE TART CANCEL 8 The LED Calibration will close 9 On the Lane Status Panel the Separate indicators will turn green Section 9 3 10 On the Protocol Status Panel the progress clock will begin and the progress block will appear Section 9 3 9 3 888 744 2244 6 www sagescience com e support sagescience com 9 3 Monitoring a Run The Lane Status Pane has three indicator lights per lane When the instrument is idle all Idle indicator lights are light gray Reference Idle Separate Elute When a protocol is running the following indicator panel will be displayed sample ID Current Elution Timer Reference Idle Separate sampoen 7 RR T I a T I O R T 00 00 00 e Green Separation If a lane is separating fragments the Separate indicator will be green e Orange Elution Ifa lane is eluting a size range the Elute indicator will be orange and the elution timer will be active Note f there is a time value in the Elution Timer field and the Separate indicator is green then the elution has been completed Pippin P
13. e USB key on the right File Manager Tab Destination Drive File RiromeippriPoonrrentogs201202 RR clas Directory Destination Drive Files UNMOUNT FLASH DRIVE File Types File Commands 16 1 888 744 2244 6 www sagescience com e support sagescience com 16 2 File Types GO TU LUGS GO TU PROTOCOLS GOTO CASSETTES There are three types of files that are stored in different directories Log Files The log file directory is accessed by pressing the GO TO LOGS button Every run on the Pippin Prep automatically saves the following files which are saved with the following convention software version year month day _ hour minute second _ user input protocol name or test type file extention e Log File This is a text txt file that contains all of the data that was collected during arun This file can be used as a diagnostic tool with Sage Science support personnel Data from the log file can be viewed in the Log Review Screen using the PC Pippin Review application or copied into a spreadsheet file to recreate the data displayed in the Pippin Prep graph images e Screen Image File An image file png of the Main Tab screen at the completion of the run is saved The log and screen image files e Continuity Test After every test an image file ong is automatically saved e Calibration Test After every test an image file ong is automatically saved In addition a screenshot may be man
14. generate any bubbles that can occlude the current path Use spare buffer that has been supplied with the cassette package or pipette from the buffer chambers 6 3 888 744 2244 6 www sagescience com e support sagescience com A Important Make sure that the pipette tips used for step 4 extend all the way to the bottom of the elution modules without sealing the elution port opening If the tips seal the port opening it will be extremely difficult or impossible to empty and refill the elution module completely Test tip fit using the empty rinse cassette supplied with the instrument Note The total volume of the elution module when filled to the top is 65ul The specified starting volume of 40ul only partially fills the module Seal the elution wells with the adhesive tape strips Tape for sealing the elution wells are supplied with cassette packaging Long elutions will cause the elution wells to overflow if not sealed Place tape over the elution wells and rub firmly to fix the tape in position For best results rub the tape at the boundary of the well with a smooth hard object such as the back end or a lab marker pen Check the buffer levels in the sample wells Sample wells should be completely filled to the top with buffer If any wells are under filled top them off with additional buffer Note The total volume of the sample well is approximately 70 ul 6 3 Continuity Test The continuity test measures the current in eac
15. sss sss sss s sss sire esen ennenen ennenen eenn ees 8 1 RUNNING A PROTOCOL iirc ate crete aE EEE 9 1 OV T AA E E E O EE A E E 9 1 STN A R a E E sean saetaesued onc suanavaauterauneoueiainecueass 9 1 MOMONDO a 19T arna E ET A E A 9 4 LOO FES L ene one ee secre cmv acne eee meee 9 6 SAMPLE COLLECTION sss sss sss ssss sese eee 10 1 VEINTE ETT 10 1 Intrinsic Sample Recovery on the Pippin Prep cceeeceeeeeeeeeneeeeeeeeeneeeeeeeeaneeeeeeeeeneeeseeeenseees 10 2 Improving Product Recovery with the Field ReverSal csccccsssssssssseeeeeesneessseeeeeesseneeeenes 10 3 Improving Product Recovery of Larger Fragments with a Surfactant Rinse ss0000 10 4 Improving Product Yield by Selecting a Wider Size RAange cceeseeeeeeeeeeeeeeeeeeeeeeeeeeeeees 10 4 WRITING AND EDITING A PROTOCOL sss sss sss sss sss cesse ess seene nenen eenn 11 1 ON ca cay eaters ao e E E a 11 1 Programming a New Protocol QUICK Guide ceeeeeeeeeeeeeeeeeeeeeseeennneeeeeeeeeeeseeeeeeoeeeseeeeneees 11 2 Selecting a Cassette Definition sss s sss esse esse sees eee 11 4 L TO la a RUN TMG sisien aa aE EE E EA aE 11 5 888 744 2244 6 www sagescience com e support sagescience com 11 5 11 6 11 7 11 8 11 9 11 10 11 11 12 13 14 15 16 16 1 16 2 17 17 1 17 2 18 19 20 21 21 1 21 2 21 3 21 4 21 5 21 6 21 7 21 7 21 9 21 8 Assigning a Reference Marker external standard scccecceee
16. system s capabilities and precautions System Overview There are three components to the Pippin Prep system Instrument The instrument contains an epifluorescence detector an electrophoresis power supply an electrode array and a Linux based single board computer The system computer is accessed by an external LCD monitor mouse and keyboard The electrode array is located in the top of the sliding instrument cover and is not user accessible Software System software allows the user to enter parameters that define the DNA fragment size ranges or DNA bands that are to be collected The software also logs fluorescence and electrical current data which may be analyzed in a review screen Gel Cassettes 5 lane disposable cassettes are manufactured by Sage Science Each cassette contains precast gel and electrophoresis buffer One lane is typically used for running a DNA size marker Size markers and sample loading solution are supplied with each cassette kit Cassettes are available in several gel concentrations and types to accommodate different size ranges or applications There are two categories of gel cassette o Dye free Cassettes The DNA size marker has been labeled with a fluorescent dye TAMRA The sample DNA is not detected by the system These cassettes are used for size selecting sheared DNA Two types of strategies are used 1 External standard a DNA marker is run in a dedicated lane to determine elution ti
17. when Elution is Completed field will automatically end a run when every lane that has a programmed elution has completed its collection Users may adjust the desired run time by referring to Section 21 where estimated run times are listed for each cassette definition Users should be aware of the following when adjusting the run time parameters e Ifaruntime is entered based on run time estimates from Section 21 make sure adequate time is added to the estimate as a safety e The Run Time field will take precedence When the run time is achieved the run will end regardless of whether the elutions are complete To end arun manually go to the Main Tab and press STOP Completed elutions may be verified in the Lane Status Panel if there is a value is in the Elution Timer field and the Separate button is lit 888 744 2244 6 www sagescience com e support sagescience com 11 5 Assigning a Reference Marker external standard 1 Determine the lane into which the reference marker will be loaded 2 Inthe protocol editor enter the reference lane designation into Ref Lane field for every cassette lane Click APPLY REFERENCE TO ALL LANES Figure 10 1 shows the proper settings for running a DNA marker in lane 1 with programmed Tight extractions in lanes 2 5 Reference Lane APPLY REFERENCE TO ALL LANES USE INTERNAL STANDARDS Tight Range Time Peak RefLlane Figure 11 1 The correct confi
18. will allow a new name for the file to be applied Protocol Changes MOT Applied amp SAVE AS All protocol files are saved in a directory home pippin Pippin Prep Protocols and may be accessed in the File Manager tab 11 11 Warnings and Indicators BP Range Flag When a Minimum Size Range has been programmed into a sample lane the BP Range Flag field for that sample will display tight and a green color BF Fange Flag 11 12 888 744 2244 6 www sagescience com e support sagescience com When a broad size cut range larger than the minimum has been programmed into a sample lane the BP Range Flag field for that sample will display broad and an orange color AP Range Flag Pause Enabled Indicator If the pause feature has been entered for a sample lane Pause On indicator will be displayed Pause On 11 13 888 744 2244 6 www sagescience com e support sagescience com Warnings Text Box If there are errors or exceptions within a programmed protocol a message will appear in the Warnings text box identifying the nature of the discrepancy Warnings Some elution ranges will cause chamber overflow Some start values missing Some end values missing some start fend values ill defined some APstart or BPthresh values out of ladder range Pippin Prep Operations Manual software v 6 13 CD9 11 14 888 744 2244 6 www sagescience com e support sagescience com 12 System
19. 6 www sagescience com 6 support sagescience com 21 4 1 5 Agarose ethidium bromide 250 bp 1 5kb Marker A Product Number CSD1510 Use Cassette Definition 1 5 Agarose Marker A DNA Marker A Typical times to detector zi l l l l l l l 30 00 40 00 50 01 00 04 10 0141 20 01 30 01 40 Time hh rmr Estimated Collection Times for Minimum Size Range Tight Targets 300 Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Specifications subject to change without notice see page 21 1 for specification definitions 21 5 888 744 2244 6 www sagescience com 6 support sagescience com 21 5 0 75 Agarose ethidium bromide 2 8kb Marker D Product Number CSD7510 Use Cassette Definition 0 75 Agarose Marker D DNA Marker D Typical times to detector kb 0 5 1 2 3 4 535 D amp 10 l l l l l l 00 30 00 40 00 50 01 00 01 10 01 20 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets Time to Time to Collect min bp Mean stdev Important Input sample loading of 0 75 gel cassettes effects accuracy In the 0 75 gel cassettes the input load has a large effect on size selection accuracy Lower input loads will size select a larger target than programmed while higher input loads will produce a smaller target than programmed Users should refer to th
20. A Marker G Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets Time to Time to Collect bp mm 100 00 30 OU 40 Time nh rmnm Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 100 600 bp Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 21 1 for specification definitions 21 10 888 744 2244 6 www sagescience com 6 support sagescience com 21 9 1 5 Agarose Dye Free 250 bp 1 5 kb Marker K internal standards Use Product Number CDF1510 Use Cassette Definition 1 5 DF Marker K DNA Marker K Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets 00 20 00 25 Time hh mm Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 250 600 bp 800 1000 bp 1200 1500 bp Sample Recovery 50 80 50 80 50 80 Specifications subject to change without notice see page 21 1 for specification definition 21 11 888 744 2244 6 www sagescience com 6 support sagescience com 21 8 2 Agarose Dye Free 100 bp 600 bp Marker E external marker Use Product Number CEF2010 Use Cassette Definition 2 EF Marker E DNA Marker E Typical times to det
21. Marker F DNA Marker F Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets Time to Time to Collect bp min 63 r e pee e 90 Time hh mm Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 90 250 bp Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 21 1 for specification definitions 21 8 888 744 2244 6 www sagescience com 6 support sagescience com 21 7 2 Agarose Dye Free 100 bp 600 bp Marker L internal standards Use Product Number CDF2010 Use Cassette Definition 2 DF Marker L DNA Marker L Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets 00 30 00 40 Time hh mm Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 90 250 bp Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 21 1 for specification definitions 21 9 888 744 2244 6 www sagescience com 6 support sagescience com 21 7 2 Agarose Dye Free 100 bp 600 bp 25 ul elution volume Marker G internal standards Use Product Number SDF2010 Use Cassette Definition 2 DF Marker G small volume elution DN
22. Pause BP Thresh Sample ID Template Sample 1 Reference ee 4 optional Important Minimum size ranges are may not be the ideal for some applications Very narrow size ranges collect less DNA than larger ranges Users should consider the requirement for distribution vs yield see sec 7 4 888 744 2244 6 www sagescience com e support sagescience com 8 Save After programming a new protocol or editing an existing one the protocol file must be saved prior to use If the protocol is new press Save If a protocol has been edited a yellow alert will be displayed in the Protocol Changes Not Applied field Save will save the file under the previously saved name and Save As will allow a new name for the file to be applied Protocol Changes NOT Applied Z SAVE AS All protocol files are saved in a directory nhome pippin Pippin Prep Protocols and may be accessed in the File Manager tab 11 8 Range Mode programming broad size range extractions 1 Assign a reference lane A DNA reference marker is required for broad range cuts Enter the reference lane number into the Ref Lane field for the sample lane 2 Select the Range programming mode Click the Range button for the lane from which the cut will be extracted 3 Enter a range values in the BP Start and BP End fields The median target value of the collection range will auto fill the BP Target field 4 Opti
23. an be used 3 If an Elution channel right column has failed continuity Replace buffer in the elution module of the failed lane and refill with 40 ul of fresh electrophoresis buffer and retest the cassette If the lane fails again do not use the lane for samples However the failed lane may be used to run an external reference marker Continuity Test Continuity Test Parameters separate max mA elute max mA 50 Test paramete rs _ gt separate min mA elute min mA 55 ree Actual Lane Currents mA Separation El PAIE do not use lane FAIL use lane for reference only or replace buffer and retest RETURN Figure 6 5 The Continuity Test screen The elution channel in lane 1 has failed Pippin Prep Operations Manual software v 6 13 CD9 6 5 l 888 744 2244 6 www sagescience com 6 support sagescience com Sample Preparation Input Sample Characteristics When running the Pippin Prep characteristics of input DNA can affect separation resolution and efficiency of product recovery The following general guidelines should be followed lonic strength The ionic strength of the sample should be lower than the ionic strength of the buffer 80mM monovalent ions High salt concentrations can result in slower than expected DNA mobility Protein in the sample DNA binding proteins such as ligases or polymerases can affect the mobility of fragments during separation Proteins can also reduce DNA
24. and T End The pause function temporarily turns off power to the electrodes and suspends run timers allowing users to remove the sample rinse the elution well if desired and then resume the instrument run to collect additional sample 11 10 888 744 2244 6 www sagescience com e support sagescience com A 4 Tight Range 6 Important A pause will require the user to manually resume the protocol from the Main Screen controller The instrument lid may be opened during the pause and sample retrieved Enter Sample ID information Time Peak RefLane BP Target BP Stat BP End BFP Pause T Start T End TPause BP Thresh Sample ID Template Save After programming a new protocol or editing an existing one the protocol file must be saved prior to use If the protocol is new press Save If a protocol has been edited a yellow alert will be displayed in the Protocol Changes Not Applied field Save will save the file under the previously saved name and Save As will allow a new name for the file to be applied Protocol Changes NOT Applied amp SAVE AS 11 1 All protocol files are saved in a directory home pippin Pippin Prep Protocols and may be accessed in the File Manager tab 10 Peak Capture Mode programming peak band collections Select a reference lane Click the Ref button for the lane into which the DNA reference marker will be loaded A DNA marker reference is required for peak
25. ation 3 2 888 744 2244 6 www sagescience com e support sagescience com Setting up the Pippin Prep Remove the accessory box located on inside edge of box at the front end of the instrument Remove the keyboard mouse and instrument power supply from their packaging Firmly grip both sides of the instrument and lift it from the foam packaging insert The Pippin Prep weighs approximately 14 lbs Place the Pippin on a table or bench top Remove the computer monitor from the manufacturer s box and connect the monitor to Pippin Prep using supplied video cable Connect monitor to power using power Supply supplied with monitor Insert USB connector from computer key board into port located in the back of the Instrument Connect mouse to Pippin Prep via USB or PS2 connector at back of instrument Connect monitor cable into the video port located in the back or the instrument Connect monitor to power using power supply and cords supplied with monitor Connect Pippin Prep instrument to power via supplied power supply and cable Power connector is at rear of instrument Press power switch located on the rear of the instrument and wait for software to launch approximately 30 seconds The Pippin Prep ts ready for use The software should automatically launch allow 30 seconds Figures 3 2 and 3 3 on the next page show the rear panel of the Pippin Prep Figure 3 4 on the following page shows the front panel 3 3 888 744 2244 6 ww
26. ccuracy may be affected by load amounts over 2 ug and the distribution profile of DNA fragments See www sagescience com support for instructions on using higher amounts of DNA Deviation of actual target value from software input value divided by the actual value 2X standard deviation of replicate samples Recovery is measured using known amounts of plasmid marker ladder mee ail ta Important DNA input samples should be purified to eliminate DNA binding proteins such as ligases polymerases restriction enzymes Bound proteins can alter mobility and reduce recovery Specifications subject to change without notice 888 744 2244 6 www sagescience com 6 support sagescience com 21 1 3 Agarose ethidium bromide 90 250 bp Marker C Product Number CSD3010 Use Cassette Definition 3 Agarose Marker C DNA Marker C Typical times to detector 20 50 F75 100 l l l 00 45 01 00 01 15 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets Time to Time to Collect min 67 82 118 Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Accuracy lt 5 Reproducibility lt 5 Specifications subject to change without notice see page 21 1 for specification definitions 21 2 888 744 2244 6 www sagescience com 6 support sagescience com 21 2 2 Agarose
27. ck the Tight button for the lane in which the tight cut will be extracted 3 Enter a value in for the BP Target This value should represent the median value for the desired size range to be collected The actual range to be collected will auto fill the BP Start and BP End fields 4 Optional Enter a BP pause value If a value is entered into the pause field the system will pause when it has estimated that that base pair value has been collected during the elution of that lane A pause will allow a user to remove two consecutive cuts from a sample The two cuts will usually have overlapping size distributions User may wish to use the pause feature for two reasons 1 to retrieve nearly identical cuts from the same sample or 2 to avoid overflow of the elution module during collections that require a long elution period The BP pause value must be within the extraction range between BP Start and BP End The pause function temporarily turns off power to the electrodes and suspends run timers allowing users to remove the sample rinse the elution well if desired and then resume the instrument run to collect additional sample Important A pause will require the user to manually resume the protocol from the Main Screen controller The instrument lid may be opened during the pause and sample retrieved 5 Enter Sample ID information Tight Range Time Peak RefLane BP Target BP Stat BP End BP Pause T Start T End T
28. dicator light will activate Press Accept to accept the new setting Instrument Name Blue 2 IOF Server Port Use Reverse Field for Sample Recovery Date Time A change in settings will not be accepted if an experiment is underway REVERT Settings Changed ACCEPT 13 2 888 744 2244 6 www sagescience com 6 support sagescience com 14 Running in Manual Mode The Pippin Prep may be run without using a programmed protocol and with manual control 1 Select the Manual Mode from the Protocol Name drop down menu in the Run File Manager Manual Mode 07 Feb 2012 17 23 08 OF Feb 2012 202307 2 Press Start on the controller 3 Sample lanes may be run individually by pressing the Separate button and elutions are carried out by pressing the Elute button After elution press the Separate button to end the elution and return to separate mode or press the Idle button to stop the run on that lane Sample ID Current Elution Timer Reference Idle Separate Elute Pippin Prep Operations Manual software v 6 13 CD9 14 1 888 744 2244 6 www sagescience com 6 support sagescience com 4 Press Pause Resume and Stop buttons are also operative as in automated runs TEST START CALIBRATE SMNAFSHOT IMFO SHITE image and when it is in position to be eluted See cassette specifications at the end of this manual to determine the best timing for manual runs
29. e Ambient temperature 17 32 C Humidity 10 80 non condensing Standard laboratory precautions should be taken when handling Pippin Prep Gel cassettes and operating the Pippin Prep e Wear a lab coat safety glasses and gloves e Use in proximity of an eye wash station and or running water Important For ethidium bromide containing gel cassettes running buffer and spare electrophoresis buffer contain 5 ug ml of Ethidium bromide Cassettes and buffer should be disposed of as laboratory waste according to institutional lab policy 2 1 888 744 2244 6 www sagescience com e support sagescience com 3 Unpacking and Installation Unpacking the Pippin Prep The Pippin Prep Instrument The Pippin Prep instrumentation is shipped in two boxes one will contain the Pippin Prep and Accessories and the second box will contain the computer monitor in the manufacturer s original packaging With the boxes in the upright position open and confirm that the following items are enclosed Monitor e LCD computer monitor e Video cable e Power cord Pippin Prep e Pippin Prep Instrument e Accessory box o Computer keyboard USB Computer mouse USB Power supply Plug adapter adapts non U S power cords see Figure 3 1 Power cord Rinse cassette for maintenance of electrodes Calibration cassette for setting optical baseline before each run O O O O GO IEC 320 C14 IEC 320 CS Figure 3 1 Adapter plug for non US power cord install
30. e hh mm HEP End P lt 2L 2 2 Figure 12 1 Control DNA profile with reference marker and DNA extraction protocol for a 2 agarose DNA control kit run 12 1 888 744 2244 6 www sagescience com 6 support sagescience com 60 Target 150 bp Ave Size 149 bp H i Conc ng ul 3 60 ESE kal iba tees a 300 400 500 700 1500 40 20 Figure 12 2 Example of a Bioanalyzer result for the 150 bp target of a Control DNA run ona 2 0 Agarose Cassette Important Data are not intended to imply guaranteed results or performance Control standards are intended to demonstrate that the Pippin Prep system is functioning as expected and that proper operational technique is being used 12 2 888 744 2244 6 www sagescience com 6 support sagescience com 13 System Options Tab The default screen on the Pippin Prep is a tabbed format The Protocol Editor is the fifth tab Overview The System Options Tab allows users to set or change global configurations for the software and Pippin Prep System Options Tab Instrument Name UDP Port Reversible ep Dan anne Field e Date and Time This screen allows users to set the following Instrument name A name can be given to or changed for the instrument that will be displayed in the Main Tab and in the log files UDP Server Port Provides and address for remote communications between the Pippin Prep and wireless control Contact Sage Science for support Reversibl
31. e Field for Sample Recovery When activated all sample collections will terminate with a 5 second field reversal to improve recovery When deactivated all sample collections will not include the field reversal The default setting is ON When activated the current will be reversed on all subsequent runs until the user changes the setting through the System Options tab Date Time This allows users to set or change the date and time settings for the instrument The date and time settings are recorded in log files and log file names 13 1 888 744 2244 6 www sagescience com 6 support sagescience com To Change the settings press the System Options Tab Instrument name 1 Enter a text into the Instrument Name field 2 The Settings Changed indicator light will activate Press Accept to accept the new setting Reverse field option 1 Press the Use Reverse Field for Sample Recovery button The indicator display will change from dark gray to white and display change from OFF to ON 2 The Settings Changed indicator light will activate Press Accept to accept the new setting Important The reverse field setting will apply to all runs until it is changed in this tab Date and time 1 Edit the date or time display directly in the Time Display field or select the calendar icon next to the field and edit the time or select a date 2 The Settings Changed in
32. e File Manager Tab The files are saved internally on the Pippin hard drive in the directory named home pippin Pippin Prep Logs in a folder with a year month YYYY MM folder name Log files names have the following convention software version year month day hour minute Second _ user input protocol name or test type file extension An example of the four types of log files and file structure in the File Manager Tab is shown below Main Protocol Editor Log Review File anager Pippin Folder d home BluePippin Logs 2012 02 Pippin Listing fone level upi P5 2012 02 15 11 28 22 LEDCalibration png P5 2012 02 15 11 28 22 ProtocolA txt PS 2012 02 15 11 28 22 ProtocolA png PS_ 2012 02 15 11 05 39 Continuity png 9 6 888 744 2244 6 www sagescience com e support sagescience com 10 Sample Collection Sample Well Elution Module _ ale puffer Buffer Chamber E l Y d Chambers Pa I a Q c a d rh T z Ti Pi gt ni S E e N i a K P lt J gt o boa OB cw 10 1 Overview 1 Samples can be removed from elution modules using a standard 100 200uI pipette Important Make sure that the pipette tips used for collection extend all the way to the bottom of the elution modules without sealing the elution port opening If the tips seal the port opening it will be extremely difficult if not impossible to completely recover the conten
33. e chart on the next page to determine how much of an offset should be expected and some method development will likely be required to achieve desired results Input amounts that are below 1ug have not fully characterized and may require method development by users Performance Specifications Accuracy Dependent on input load See chart on following page Specifications subject to change without notice see page 21 1 for specification definitions 21 6 888 744 2244 6 www sagescience com 6 support sagescience com Input load dependence of 0 75 Pippin Prep cassette 10000 5 i i o lug _ 2000 L Haug Q mi Sug AT Kere 10ug TA SES 6000 m co Q 5 5000 HHHH 9 S Q 4000 in i l j 9 gt 3000 7 775 L 1000 T s T 4 L L 1000 2000 3000 4000 5000 6000 7000 8000 9000 Programmed Bptarget size bp To use this chart Find the desired target size on the y axis and follow the value to the approximate input amount Enter the corresponding x axis value into the protocol editor software Each point is an average of four experiments Error bars for the 5 ug load represents two standard deviations 888 744 2244 6 www sagescience com 6 support sagescience com 21 6 3 Agarose Dye Free 90 bp 250 bp Marker F internal standards Use Product Number CDF3010 Use Cassette Definition 3 DF
34. e very narrow size distributions from sheared genomic DNA However narrower size distributions will necessarily mean that a smaller fraction of the inout DNA will be recovered Users should broaden their collection ranges if the default tight settings do not produce enough DNA for their application See Section 10 5 for details Collection ranges 7 10 5 CV 6 6 CV l input gDNA 5 ug 5 0 CV Tight 888 744 2244 www sagescience com 6 support sagescience com DNA undergoing elution is smaller than DNA at the detector The branch point between the separation and elution channels is downstream from the detector position Figure 4 2 shows a cassette channel to illustrate During normal operation the leading edge of the DNA fraction scheduled for elution passes the detector before the start of elution by up to several minutes This offset can give rise to the impression that sample elution is late even in runs that are functioning properly LED Detector Branch Point DNA Migration OO Figure 4 2 An illustration of the time and base pair difference between the detector and branch point The rate of electrophoresis is faster in the separation channel than in the elution channel This may cause misconceptions with regard to the timing of broad size selections For instance if one sample is programmed to select from 400 600bp and a second sample is programmed to select from 200 600 bp the narrower range will f
35. ector 70 75 150 300 S JY l 7 sa l Si l l i l 00 30 00 40 00 50 01 00 0141 10 01 20 0141 30 01 40 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets Time to Time to Collect bp min 150 51 w o o Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 100 600 bp Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 21 1 for specification definitions 21 12
36. eeeeeeeeeeenseeeeeeeeeseeeeenenneeeenens 11 6 Assigning Reference Markers internal Standards cccssssecsseesseeesesseeeeenseesesseeeeeeneessoeas 11 7 Programming a Tign CONCCUION ii E a Ea a Aaa 11 8 Range Mode programming broad size range extractions sssssessssssssesssssssseeeeeessssseeeeeeessss ess 11 9 Time Mode programming timed CGULIS ssssssssssssssssssssssseeeseesssseeeennee ennenen 11 10 Peak Capture Mode programming peak band collections ssssssnnssnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 11 11 Warnings ANG INGICALON S sisri aaa aaa 11 12 SYSTEM VALIDA HON erene 12 1 SYSTEM OPTONS TAB H 13 1 RUNNING IN MANUAL MODE sssss sss sss sss ssss sss sss essen enen nenen eenn 14 1 ANALYZING RUNS LOG REVIEW TAB sss sss sss sss sss ces sese e nenen eenn 15 1 MANAGING FILES FILE MANAGER TAB cccescceseeceeeeeeseenseeeeeseneees 16 1 OV NN a tp ees a ee sed ace een sees ree du mee ieee Ras s 16 1 Fe OS Sioa acc carci cece eee ene ates decieaeenete conemeseeeeeenscetneceucsueenstelamecevsmeaeneatenseies 16 2 UPGRADING SOFTWARE ss sss sss sss sss ss esse ee nenen enen 17 1 Extracting the Files to a USB flash drive csssssssssssssssssssssessssseeenne ennenen ennenen 17 1 Upgrading the Pippin Instrument Softwar e eseecceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeees 17 3 WARRANTY AND SERVICE ss sssssss sese sees enen 18 1 ORDERING INFORMATION sss sss sss ssc ss sss e seene enne
37. fault screen on the Pippin Prep is a tabbed format The Main Tab is the first tab and the default screen 9 1 Overview The Main Tab is the screen with which users load protocols run the instrument and monitor optical signal and electrophoresis Main Tab Protocol Editor File Manager System Options Lane Sample ID Current Elution Timer Reference Idle Separate Elute Status Graph S mn E TH mR P Panel Display J ooo Fooco00 f 4 Toco DIT fs fl S O fc CT J ia S O ee Co Fon LR ft a Z Protocol 00 00 01 00 02 00 03 00 M 05 00 06 00 07 00 08 00 Statu S Image se Panel Display eer emo ccess Control Panel TEST START ae CALIBRATE SNAPSHOT INFO SHUTDOWN 1 1 1 1 1 1 1 1 1 00 00 01 00 02 00 03 00 04 00 05 00 06 00 07 00 08 00 Time hh mm 9 2 Starting a Run 1 Go to the Main Tab 2 If necessary select the protocol to be run from the Protocol Name drop down menu in the Protocol Status Panel The last protocol that has been programmed will automatically be loaded See Chapter 11 Writing and Editing a Protocol for instructions on programming Protocol Name protocol xyz Progress hh mm ss Voltage Clock l 99 9 l 07 Feb 2012 17 07 31 Remote Access Estimated Completion Time l 07 Feb 2012 18 06 49 Note The instrument optical array must be calibrated daily before running samples A calibration cassette is provided with each Pippin Prep instrument 888 744 2244 6 www sage
38. g located in the System Options Tab of the Pippin Software The use of this option is recommended but may turned off by users Figure 10 3 below shows the improvement that can be achieved Figure 10 4 shows the setting option in software When activated the setting will be applied all subsequent runs until the user again changes the settings in the System Options Tab 2 cassette pBR 322 Mspl digest 40 min elution With current reversal o AAT ooo nda Ss ee Without current reversal 200 300 400 500 Size of Fragment Bp Figure 10 2 Improving recovery with a 5 sec current reversal A 40 minute elution of a plasmid restriction digest was run and each data point represents an individual band Instrument Mame P00 WIDP Server Port Lise Reverse Field for Sample Recovery Use this button to turn field reversal OFF ON and then press Accept The default setting is ON Figure 10 3 System Options Tab with expanded view of Reverse Field Option 10 3 888 744 2244 6 www sagescience com e support sagescience com 10 4 Improving Product Recovery of Larger Fragments gt 500bp with a Surfactant Tween Rinse Sample recoveries of targets over 500 bp can be improved by using a surfactant rinse Remove the sample from the elution well at the end of the run Add 40ul of Tween solution Supplied with the cassettes to the elution well Wait 1 minute Remove solution and pool with the ori
39. ge Science 500 Cummings Center Suite 3150 Beverly MA 01915 USA suppot sagescience com Instrument Name Mario Instrument ID s Version 4 70 Build Date 31 Jan 2012 FV Version 3 12 IP Address 127 0 0 1 MAC Address 00 30 18 AB D9 62 UDP Server Port 50000 SOFTWARE UPGRADE G 2011 Sage Science http sagescience com Parts 2011 Solidus Integration http solidusintegration com Parts 2011 National Instruments http ni com 4 Press OK at the warning prompt the Software Upgrade Window will launch Warning SELECT OK TO EXIT THE MAIN PROGRAM AND LAUNCH UPGRADE UTILITY Ta BD N fa i a A a AN a N AA CX V j Ar La ava fi Lem Wi Be 4 3 A A C Pippin Prep Operations Manual software v 6 13 CD9 17 3 qQ 888 744 2244 6 www sagescience com e support sagescience com 5 Press OK at the warning prompt the Software Upgrade Window will launch Current Version Hard Drive New Version Flash Drive 16 Jul 2010 13 00 a 14 Oct 2010 13 00 3 60 16 Jul 2010 15 35 O oo 14 Oct 2010 16 15 T DBA1E0D753B44069 EE1CB198F A72DB25 is S grade rs to make upgrade IS eu EOZ ect INSTALL eee pn nea RT a MOUNT FLASH DRIVE to rem a afely INSTALL UPGRADE UNMOUNT FLASH DRIVE 6 Press INSTALL UPGRADE this will take about 5 seconds INSTALL UPGRACE UNMOUNT FLASH DRIVE 7 Press UNMOUNT FLASH DRIVE UNMOUNT FLASH DRIVE Warning Be sure to Unmount
40. ginal extracted sample 10 5 Improving Product Yield by Selecting a Wider Size Range The Pippin Prep can produce very narrow size distributions from sheared genomic DNA However narrower size distributions will necessarily mean that a smaller fraction of the input DNA will be recovered Users should broaden their collection ranges if the default tight settings do not produce enough DNA for their application Figure 10 4 shows an example of the effect of size range selection vs yield Bioanalyzer Result Average 3P End Size bp BP Start BP End Figure 10 4 Increasing yield by widening the size selection range 10 4 888 744 2244 6 www sagescience com e support sagescience com 11 Cassette Type Selector Protocol Parameters File Commands af Writing and Editing a Protocol The default screen on the Pippin Prep is a tabbed format The Protocol Editor is the second tab 11 1 Overview The protocol editor tab is the screen with which users create new protocols or edit existing protocols for DNA size selection A protocol is created for a cassette type within the applicable target range and is saved as a named protocol file with a ppprot file extension When running a cassette an appropriate protocol file is selected and applied to the run A protocol file may be applied to any run provided the cassette type is correct Protocol Editor Tab Protocol Name Run Time Selector Warnings Text Bo
41. guration for a protocol with the DNA marker in lane 1 The values may be entered with a single step 888 744 2244 6 www sagescience com e support sagescience com 11 6 Assigning Reference Markers internal standards 1 Markers must be loaded into every lane with the sample 30 ul of sample with 10 ul of internal standard loading solution 2 Inthe protocol editor press USE INTERNAL STANDARDS This will auto fill the Ref Lane fields with reference marker internal to the sample The software will determine the timing of elution based on the migration of the DNA marker within its own lane 3 Figure 11 2 shows the correct assignation of marker lanes for internal standard runs Reference Lane APPLY REFERENCE TO ALL LANES USE INTERNAL STANDARDS Tight Range Time Peak Ref Lane Figure 11 2 The correct configuration for a protocol using internal standards T Important When using internal standards all samples must be prepared the Pippin Prep internal standard loading solution which ts formulated for the cassette type and size range to be used The internal standard loading solution contains the markers 888 744 2244 6 www sagescience com e support sagescience com 11 7 Programming a Tight Collection 1 Assign a reference lane A DNA reference marker is required for tight cuts Enter the reference lane number into the Ref Lane field for the sample lane 2 Select the Tight programming mode Cli
42. h separation and elution channel and determines whether they are within the expected values for a successful run Important Temperature affects electrical current readings If cassettes have been refrigerated they will fail the current test until the cassette is at least 17 C 62 F Should this be the case wait until the cassette temperature has equilibrated to room temperature and re test Press Test With the cassette in the optical nest close the lid and press TEST on the controller on the Main Tab TEST START CALIBRATE SHAP SHOT INFO SHUTO GA 6 4 888 744 2244 6 www sagescience com e support sagescience com 2 Continuity Test will Automatically Run The continuity test screen will launch The separation and elution channel test parameters are displayed and the electrical current results will be listed after several seconds If the test returns a PASS message press RETURN and continue to baseline calibration Continuity Test Continuity Test Parameters separate max mA elute max mA separate min mA elute min mA Actual Lane Currents mA Separation Elution RETURN Failed Continuity Tests 1 A failed test is indicated by a FAIL message and the failed channel is highlighted in orange Figure 6 5 shows a failed test screen elution channel in lane 1 2 If a Separation lane has failed left column continuity Do not use that lane Remaining passing lanes c
43. ic effect on a DNA sample when a sample well in not completely filled Pippin Prep Operations Manual software v 6 13 CD9 8 1 888 744 2244 6 www sagescience com e support sagescience com Re check the buffer level in the sample wells Make sure that sample wells are completely full to the top with electrophoresis buffer Top off with additional buffer if necessary The total volume of the sample well is 70 ul Remove 40ul of buffer from the first sample well and load 40ul of sample or marker into that well Take care not pierce the agarose with the pipette tip There is gel on all sides and bottom of the sample well In addition there is an agarose chimney surrounding the top of the sample well that protrudes up through the cassette cover see Figure 10 When removing buffer some users find it useful to immerse the pipette tip just below the surface of the buffer and follow the liquid level down with the tip as the buffer is removed When buffer removal is completed there will be 30ul of buffer left in the well When adding sample place tip of pipette just below the surface of the buffer and follow the liquid level up with the tip as the well fills Don t be concerned if the sample well slightly overfills The density of the sample will allow it to sink before it can flow out of the well 3 Repeat step 2 for remaining wells 8 2 888 744 2244 6 www sagescience com e support sagescience com 9 Running a Protocol The de
44. iding cover Information Electrophoresis buffer is 50 mM Tris 30 mM TAPS 0 1 mM EDTA In cassettes with Ethidium bromide the concentration is 5 ug ml During elution EDTA does not rapidly equilibrate across the ultrafiltration membrane of the elution module and so the final concentration of EDTA in eluted samples is elevated to 1 2 mM Information E uted samples can be used directly for ligation and amplification without buffer exchange 10 2 Intrinsic Sample Recovery on the Pippin Prep Intrinsic DNA recovery in Pippin Prep cassettes is determined by running known amounts of a plasmid restriction digest on the Pippin Prep collecting a broad range of fragments from the digest and comparing the input and product profile quantitatively using the Agilent Bioanlyzer 2100 The intrinsic recovery of DNA fragments on all Pippin cassette types is between 50 80 Figure 10 1 below shows the recovery of a 5kb band from a 0 75 cassette 59 Recovery Blue input DNA1 2 dilution Red extracted DNA 1 1 dilution N P U an Bioanalyzer Standard Figure 10 1 An example of the intrinsic recovery of 5 kb band on a 0 75 agarose cassette 10 2 888 744 2244 6 www sagescience com e support sagescience com 10 3 Improving Product Recovery with the Field Reversal The Pippin Prep provides a brief current reversal at the end of a sample elution can improve sample recovery by up to 20 The field reversal is a default settin
45. inish eluting before the broader one even though both elutions complete collection at 600 bp Rate of Migration Separation 25 Faster Elution Figure 4 3 An illustration comparing the relative rate of electrophoresis in the separation and elution channels 4 2 888 744 2244 6 www sagescience com e support sagescience com Elution creates a jump in the optical background of the sample lanes Since ethidium bromide is positively charged it will migrate toward the electrode during electrophoresis moving in the opposite direction from the DNA During the initial phase of a Pippin Prep run the separation channel will become partially depleted of ethidium bromide However when elution begins a front of fresh ethidium bromide from the unused elution channel will migrate up through the separation past the detector and create a jump in the ethidium bromide background signal which can be misinterpreted as a DNA signal This is illustrated in Figure 4 4 The ethidium bromide front passes the detector approximately 4 5 minutes after start of elution The ethidium bromide front may not be noticeable when DNA input is high Ethidium Migration 28 DNA Migration _ __ Elution Channel 0000 0010 00 20 00 30 0040 0050 O100 O110 120 orso orso orso ozo Ethidium bromide fronts Time hh mm due to elution 00 00 00 10 00 20 00 30 00 40 00 50 01 00 01510 01 20 01 30 01 40 01 50 02 00 Time hh mm Figure 4 4
46. ive Flash drive Pippin to flash drive prior to flash drive to Pippin removal A N Warning Be sure to Unmount Flash Drive prior to removal Failure to do so may cause data loss Failure to do so may cause data loss and problems with future upgrade procedures 16 3 888 744 2244 6 www sagescience com 6 Upgrading Software 17 17 1 Pippin upgrade software is available for download at support sagescience com www sagescience com support The files are provided in a zip file and the contents must be extracted to the root directory of a USB flash drive Extracting the Files to a USB flash drive Download the Software Upgrade package by pressing the appropriate link on www sagescience com support in the Downloads section under Software Press Save File when prompted The zip file will likely save to a Downloads folder on your computer The file will be named pippinupgrade version number zip Copy the zip file to a USB drive Make sure the file is transferred to the root directory and not placed within a folder Right click on the zip file and select Extract All Name Ef pippinupgraded Fa Fa Open Open in new window Extract All Scan for Viruses Move to Quarantine Open with Adobe Drive C54 Send to Copy Create shortcut Delete Rename Date mod 2 E and F 17 1 888 744 2244 6 www sagescience com e sup
47. l be displayed in the Protocol Changes Not Applied field Save will save the file under the previously saved name and Save As will allow a new name for the file to be applied Protocol Changes MOT Applied SAVE AS All protocol files are saved in a directory home pippin Pippin Prep Protocols and may be accessed in the File Manager tab 11 9 Time Mode programming timed cuts Important The time mode does not reference a standard Cassette to cassette variablity and environmental running conditions will cause a wide variation in selection accuracy 1 Select the Time programming mode Click the Time button for the lane from which the cut will be extracted A DNA marker reference standard is not required 2 Enter atime values in the T Start and T End fields Sample elutions will occur at exactly those times after the start of the run 3 Optional Enter a Time pause value If a value is entered into the pause field the system will pause at that time after the start of a run A pause will allow a user to remove two consecutive cuts from a sample The two cuts will usually have overlapping size distributions User may wish to use the pause feature for two reasons 1 to retrieve nearly identical cuts from the same sample or 2 to avoid overflow of the elution module during collections that require a long elution period The T pause value must be within the extraction range between T Start
48. lect a log txt file Select Pippin log file Cancel 2010 10 26 09 39 06 _PP Logit sts Help Pippin Prep Operations Manual software v 6 13 CD9 15 2 888 744 2244 6 www sagescience com 6 support sagescience com 4 This will populate the Review Log Screen with data from the selected run log Jhome pippin PippinPrep Logs 201 1 05 2011 05 24_10 25 26_tests_2 1 88tight tt Protocol Name Cassette Name Un Time hhimm tests 2 188tight 2 Marker B Off BP Target BP Stat BP End BP Pause Star ne T Pause BP Thresh Sample ID Template Sig Mon gt Range Flag ause Enabled D rotocol Information DNA Marker Times and Values TELELE es i yy D Peaks Found ANALYZE PEAKS SNAPSHOT Optical Electrophoretic S Se Current Traces igna Current drops Traces during elution Visualizing Pippin Run Data The current and optical signal traces are color coded and a key is displayed to the right of the Current graph A check mark next to each lane will toggle the visualization of that lane on and off The type and color of the lines may also be adjusted by clicking inside the line field and holding the left click on the mouse The same styles are applied to both the signal and current traces Lane 1 iph S Turn line Lane 2 Iph visualization on off Lane 3 Iph Lane 4 Iph Lane 5 Iph Hold left click on Filtered Ref mouse to change line color or style Threshold Ee Egeu EE D A i i k i IFA
49. ming Capacity is 4 samples cassette 2 Internal standards DNA markers are added to each sample to determine elution timing Capacity is 5 samples cassette o Cassettes with intercalating dye ethidium bromide The agarose gel and running buffer contains ethidium bromide Ethidium bromide is an intercalating dye which allows the system to detect DNA above a certain threshold These cassettes can be used for applications in which the detection of DNA bands PCR products or restriction digests are collected band capture protocols 1 1 888 744 2244 6 www sagescience com e support sagescience com Safety and Precautions Icons Used In this manual the following icons will be used to provide the user with information pertinent to the use of the Pippin Prep Caution Warns the user that injury or instrument damage may occur if the contents of the warning are not properly followed warning are not properly followed Important Provide important information about the proper use of the system that may influence the quality of the result A High Voltage Warns of the risk of electrical shock if the contents of the O Information Provides additional information regarding the function of the system or applications for which is used Safe Use Guidelines The Pippin Prep system is designed to operate under the following environmental conditions Pollution Degree 2 Installation category 2 Altitude 2000m Indoor us
50. ncludes monitor keypad mouse and accessories BLUOOO1 Gel Cassettes with Ethidium Bromide with External Marker 10 pk External Catalogue Marker No 3 agarose with EtBr 90 250 bp CSD3010 Dye Free Gel Cassettes with Internal Standards 10 pk Catalogue Standards No 3 agarose dye free 90 250 bp CDF3010 mm 100 680000 Dye Free Gel Cassettes with External Marker 10 pk External Catalogue No Marker 2 agarose 100 600 bp CEF2010 These cassettes were designed for use with EpiCentre s Script Seq kits The elution volume in other cassettes are 40ul 19 1 888 744 2244 6 www sagescience com 6 support sagescience com Ordering Information Cont Control DNA for Performance Validation Catalogue No Control DNA for 3 Gel Cassette Int Std 90 250 bp 5 ug load 20 loads CIS3004 Control DNA for 3 Gel Cassette Ext Mrk 90 250 bp 5 ug load 16 loads CON3004 Control DNA 2 Gel Cassette Int Std 100 600 bp 5 ug load 20 loads CIS2004 Control DNA for 2 Gel Cassette Ext Mkr 100 600 bp 5 ug load 16 loads CON2004 Control DNA for 1 5 Gel Cassette Int Std 250 1500 bp 2 ug load 20 loads CIS1504 Control DNA for 1 5 Gel Cassette Ext Mkr 250 1500 bp 2 ug load 16 loads CON1504 Control DNA for 0 75 Gel Cassette Ext Mkr 1 10 kb 5 ug load 16 loads CON7504 Control DNA for 1 5 Band Capture Ext Mkr 1 kb fragment 5 ug load 4 loads CBC1501
51. nd accuracy the sample should travel through the central section of the gel column and should be bounded on all four sides by uniformly conductive media either gel or electrophoresis buffer The goal of the loading procedure is to produce this geometry in the sample loading well as illustrated in the bottom section of Figure 6 1 Properly prepared samples will be 40 ul in total volume consisting of 30 ul of DNA mixed with 10 u of Pippin Prep loading solution The loading solution contains concentrated Ficoll as a densifying agent see following chapter on Sample Preparation for details and therefore the samples will sink and form a high density layer beneath the electrophoresis buffer when pipetted slowly into the sample wells If there is insufficient conductive buffer over the sample the electrophoretic forces lines will curve upward as the sample exits the well see Figure 6 1 upper section and the sample will be drawn to the top of the cassette where it can travel out of the gel into the gap between the gel column and the plastic top of the channel Sample moving in this gap will travel at a different rate than the sample inside the gel column and will lead to elution of undesired size fractions in the eluted material In such cases the contaminating DNA will usually but not always be higher in molecular weight than the selected DNA Sample 40 tl Buffer 30 ul Sample 40 ul Figure 6 1 An illustration of the electrophoret
52. nen ennenen eenn 19 1 INSTRUMENT SPECIFICATIONS sss sss css sss cesses cesse nenen enen enen 20 1 SPECIFICATIONS FOR GEL CASSETTES c css sese seene 21 1 3 Agarose ethidium bromide 90 250 bp Marker C sssssssssssssssssessseese neee neee 21 2 2 Agarose ethidium bromide 100 600 bp Marker B ssseesssseeceneeeeeseeseeneeseaees 21 3 2 Agarose EtBr 25 ul elution well 50 600 bp Marker S for EpiCentre ScriptSeq v2 21 4 1 5 Agarose ethidium bromide 250 bp 1 5kb Marker A ccsssseeccseseeeeesnseeeeeneeeees 21 5 0 75 Agarose ethidium bromide 2 8kb Marker D ccccceeseeeeeeeeeeeseeeeneeseeneeseeneees 21 6 3 Agarose Dye Free 90 bp 250 bp Marker F internal standards scsceeseeeeseees 21 8 2 Agarose Dye Free 100 bp 600 bp Marker L internal standards ssseccssseeeeees 21 9 2 Agarose Dye Free 100 bp 600 bp 25 ul elution vol Marker G int stds 21 10 1 5 Agarose Dye Free 250 bp 1 5 kb Marker K internal standards 000 21 11 2 Agarose Dye Free 100 bp 600 bp Marker E external marker sss s esse see see 21 12 1 888 744 2244 www sagescience com 6 support sagescience com Introduction Thank you for purchasing the Pippin Prep from Sage Science The Pippin Prep is an automated preparative gel electrophoresis system We urge you to read this manual to familiarize yourself with the
53. olution should be stored at 4 C Loading solution is viscous should be equilibrated at room temperature prior to use Important Cassette should be stored at room temperature in the sealed foil packaging They are light sensitive and should not be removed until prior to use Shelf life is one year 3 6 888 744 2244 6 www sagescience com e support sagescience com 4 Achieving Best Results from the Pippin Prep The Pippin Prep extracts narrow or wide DNA size distributions more reproducibly and with higher yield than manual preparative gel techniques However due to the unique design of the pre cast gel cassette there are key differences between running the Pippin and other agarose gel methods Successful Operation It is recommended that users closely follow the instructions outlined in the following sections Section 5 8 until they become thoroughly familiar with the system The Quick Guide included with cassettes provides protocol reminders but is not intended for beginning users The keys to successful operation are Optical Calibration Section 5 Preparation of the cassettes Section 6 Purity of the input sample See Section 7 Sample loading See Section 8 Common Misconceptions Users should be aware of the following characteristics of the Pippin Prep System These are part of normal operation of the system but may seem counterintuitive at first Narrowest Is not always the best The Pippin Prep can produc
54. onal Enter a BP pause value If a value is entered into the pause field the system will pause when it has estimated that that base pair value has been collected during the elution of that lane A pause will allow a user to remove two consecutive cuts from a sample The two cuts will usually have overlapping size distributions User may wish to use the pause feature for two reasons 1 to retrieve nearly identical cuts from the same sample or 2 to avoid overflow of the elution module during collections that require a long elution period The BP pause value must be within the extraction range between BP Start and BP End The pause function temporarily turns off power to the electrodes and suspends run timers allowing users to remove the sample rinse the elution well if desired and then resume the instrument run to collect additional sample Important A pause will require the user to manually resume the protocol from the Main Screen controller The instrument lid may be opened during the pause and sample retrieved 888 744 2244 6 www sagescience com e support sagescience com 5 Enter Sample ID information Tight Range Time Peak Ref Lane BP Target BP Stat BFP End BP Pause T Start T End T Pause BP Thresh Sample ID Template Sample 1 6 Save After programming a new protocol or editing an existing one the protocol file must be saved prior to use If the protocol is new press Save If a protocol has been edited a yellow alert wil
55. p contains high voltage apparatus The instrument should be repaired by authorized personnel only Do not disassemble under any circumstance Cassettes Cassettes have a 1 year warranty If a cassette or sample lane is defective Sage Science will replace the cassette at no cost The defective cassette should be disposed of as per regulations at the user s facility Sage Science support staff may ask for a log file or image file to evaluate or troubleshoot cassette issues Maintenance lt is possible that salts will accumulate on the electrodes that are housed in the Pippin Prep lid assembly This may alter the performance of the instrument over time It is recommended that the electrodes undergo periodic rinsing To rinse the electrodes use the rinse cassette provided in the instrument shipment fill with deionized water load on the sample tray and close the Pippin Prep lid A quick rinse for several second is sufficient lf a rinse cassette is lost or damaged contact Sage Science support for a replacement No other maintenance activity is required Cassette Disposal Running buffer should be disposed of according to internal lab safety policy Running buffer contains up to 5 ug ml of Ethidium bromide Cassettes should be disposed of as laboratory waste O Note Dye free cassettes TAMRA dye bound to the reference marker 18 1 888 744 2244 6 www sagescience com 6 support sagescience com 19 Ordering Information Pippin Prep I
56. port sagescience com 5 A window which will launch to prompt you to select a destination where to extract files The prompt will suggest creating folder based on the zip file name Delete the destination folder name so that only the root directory appears E in the example below Ww 4 Extract Compressed Zipped Folders 4 Extract Compressed Zipped Folders Select a Destination and Extract Files ae E Select a Destination and Extract Files Files will be extracted to this folder Files will be extracted to this folder E pippinupgrade463 Show extracted files when complete V Show extracted files when complete Correct Files will be extracted to this folder Not Correct Files will be extracted to this folder 6 The final USB file structure should be as shown The zip file may be deleted a Removable Disk E di PippinUpgrade L Bin di Cassettes di Upgrade 4 pippinupgrade463 pin Prep Operations Manual software v 6 13 CD9 17 2 888 744 2244 6 www sagescience com e support sagescience com 17 2 Upgrading the Pippin Instrument Software 1 Insert the USB drive with the upgrade folder into the USB port on the front panel of Pippin instrument 2 From the Main Tab press INFO This will launch the information window TEST START CALIBRATE SMAPSHOT IMFO SHIITE Gi 3 From the Information Window press SOFTWARE UPGRADE Sa
57. puffer D Buffer a Chamber E Q a lls Chambers 4 U L f lt RL i D ee L T r A ee J M 1 K f i E Ld A L and p w Fi H 7 Check behind elution modules for bubbles Tilt cassette to clear Figure 6 4 A schematic of a Gel Cassette 2 Place Cassette into the Pippin optical nest The cassette should be placed into the nest in the orientation shown in Figure 6 4 above with the sample wells to the left side of the nest When inserting the cassette into the nest keep the buffer chambers tilted down so that the bubbles in the elution reservoirs won t be trapped behind the elution modules T Important Be sure the cassette is fully seated into the bottom of the nest Detection of DNA within the cassette will fail if the bottom of the cassette is not properly seated against the optical region 3 Remove adhesive strips from cassette Place one hand on the cassette and hold it firmly in the nest Grab the white tabs of the tape and pull the strips firmly and slowly toward the front of the Pippin Prep until they are removed 4 Remove buffer from elution modules and replace with 40ul of fresh electrophoresis buffer Replenishing the buffer ensures proper electrophoretic continuity When refilling empty modules place tip all the way into the module and slowly fill from the bottom withdrawing the pipette tip while dispensing and taking care not to
58. recovery from the elution module by increasing the binding of DNA to the ultrafiltration membrane at the back of the elution module For best results samples should be de proteinized prior to loading whenever possible Input DNA size distribution A knowledge of the input size distribution is obviously important to program accurate size selection settings Pippin Prep cassettes are calibrated using the Agilent Bioanalyzer to evaluate input and product sizes and so for best results input size distributions should be evaluated using the Bioanalyzer For low concentration samples the Agilent HS chip is very useful see Technote 4 on the Sage website Preparing DNA Samples for the Pippin Prep sek Bring DNA sample up to 30ul with TE Bring loading solution to room temperature For each sample combine 30ul of DNA sample with 10ul of loading solution Mix samples thoroughly vortex mixer Briefly centrifuge to collect Recommended sample Load Guidelines Maximum Load 10ug sheared genomic DNA 4 wg restriction PCR fragment Minimum Load low single nanograms Optical Sensitivity approx 200ng sheared genomic DNA ethidium bromide cassettes approx 10 15ng single bands restriction fragments PCR products or synthetic DNA fragments 7 1 888 744 2244 6 www sagescience com e support sagescience com 8 Loading Samples Proper sample loading is critical for best performance of Pippin Prep cassettes For maximum reproducibility a
59. rep Operations Manual software v 613 CD9 9 4 888 744 2244 6 www sagescience com e support sagescience com The Elution Timer records the length of time of the elution When elution is complete electrophoretic path will switch back to the separation channel and the elution timer will show the value of the total elapsed elution time Elution Timer Reference Idle Separate Elute Run progress is monitored in the Protocol Status panel The percent complete is indicated by a progress bar The Clock fields displays the current time and date The Estimated Completion Time displays the estimated time of protocol completion This value is updated and more accurate after DNA markers have been detected The Remote Access indicator is activated if the instrument is being remotely controlled from a PC via VNC server Contact Sage Science for instructions on remote control Protocol A 24 Feb 2012 05 41 52 24 Feb 2012 05 41 45 Pippin Prep Operations Manual software v 613 CD9 9 5 888 744 2244 6 www sagescience com e support sagescience com 9 4 Log Files At the end of every run the Pippin Prep will automatically save a log file txt which can be viewed on the Log Review Tab or the PC software version of Log Review A screen image png of the Main Tab is saved after the run has been finished Screen images of all continuity tests and calibration tests are also automatically saved All files may be accessed from th
60. science com e support sagescience com 3 A properly prepared cassette Chapter 6 with loaded samples should be in the optical nest Typically users will load samples while the cassette is on the nest see Chapter 8 Ensure that adhesive tape has been placed over the elution wells Press START on the Control Panel TEST START CALIBRATE SMAP SHOT Nwo SHUTDOWN An LED Calibration window will launch see Chapter 5 for details Important The Pippin Prep optical system should be calibrated for the specific type of cassette to be used dye free and ethidium bromide containing The instrument is supplied with a calibration fixture that fits into the optical nest If a lab is using only one type of cassette the optics should be calibrated daily when the instrument is in use If users switch cassette type that is to be run e g run a dye free cassette and then a ethidium bromide cassette the optics should always be re calibrated prior to running the new type If the instrument optics have not yet been calibrated or if the a new cassette type is being run a remove the cassette b and place the calibration fixture into the optical nest c select the cassette type d press CALIBRATE e The LED photocurrents will automatically adjust to a factory setting and the Calibration Status field will contain the message Calibration OK 9 2 888 744 2244 6 www sagescience com e support sagescience
61. size based fractionations tight or bp range Important f a bubble is observed do not use the lane to run the reference marker or for peak modes Important Flat bubbles between the top surface of the gel column and the plastic top of the channel will NOT affect run quality or optical detection They may be used for markers or sample without any adverse effects Agarose Delamination Optical Region Do not use this lane for DNA marker Figure 6 2 Bubble in optical path Do not use the lane for the DNA reference marker Sample Well Elution Well Buffer k l meran 9 Chamber S i _ w ct Buffer Chambers E A b uC Optical Detection gum Region Figure 6 3 The region of optical detection RP IO ARAA fa J a a AV a d L J _ k V x JM x l M N L VT PP U A Cy s e PN N LU f A M d Pa LJ h h rp Ay LU Dinnin Pran C naratinne Ip U II l FITC U VI U eld UO ak 888 744 2244 6 www sagescience com e support sagescience com 6 2 Prepare the Cassette for Loading 1 Dislodge bubbles from behind the elution wells Return the cassette to the right side up position Tilt the cassette sample well side down to release any trapped bubbles behind the elution modules into the buffer chambers Gently tap the cassette if necessary Figure 6 4 below shows location to check for bubbles Sample Well Elution Module _ m
62. t the electrophoretic current profile of a Pippin run from a run log The colored horizontal traces display the actual current signal results in mA The timing of an elution switch from separation to elution is accompanied by a 0 5 mA drop in current a CURRENT a ami 4 50 4 00 3 50 3 00 2 50 2 00 Lane Current mA T307 1 00 0 50 0 00 l l l l l 01 05 48 01 08 00 01 10 00 01 13 02 Time hhimim ss There are three icons in the upper right hand corner of the graph The functions are listed below Revert to default Grab and move view screen Zoom hold left click for Saving a Review Image An image from the review screen may be captured and automatically saved by pressing the Snapshot button The png file will date and time stamped and saved in the Pippin log file directory ANALYZE PEAKS SMAP SHOT 15 5 888 744 2244 6 www sagescience com e support sagescience com 16 Pippin File Directory Pippin Files Managing Files File Manager Tab The default screen on the Pippin Prep is a tabbed format The File Manager Tab is the fourth tab 16 1 Overview The File Manager Tab allows users to access copy and delete Pippin Prep files There are three types of files used by the system log text and image protocol and cassette type The screen is divided into two sub screens the Pippin file directory on the left anda target directory portable flash media i
63. ts of the elution module Test tip fit using the empty rinse cassette supplied with the instrument 2 Samples from standard cassettes without sealed elution modules will be recovered in 40 65 ul of electrophoresis buffer depending on the length of the elution step Samples from cassettes with tape sealed elution modules will be recovered in a fixed volume of 40 ul 3 When all samples are collected remove used cassette from instrument and dispose of it properly 4 Additional DNA product can be washed from the elution modules with a nonionic detergent After the initial product is removed and saved add 40 ul of TE 0 1 Tween 20 to the elution module After a brief incubation 1 min remove the TE Tween solution and combine it with the initial product In some cases DNA gt 4kb DNA recovery from the Pippin Prep can be doubled by this procedure Tween wash is especially important for achieving a good recovery of samples that have not been deproteinized 10 1 888 744 2244 6 www sagescience com e support sagescience com eo Ob Important Eluted samples should not be left in the cassette for longer than a couple of hours to avoid poor recovery due to adsorption or diffusion Important Used cassettes should never be allowed to sit in closed instruments for long periods of time e g overnight Under those conditions humidity from the cassette reservoirs can accelerate corrosion of the electrode assembly located in the sl
64. ually saved as at any time during a run as a png file by pressing Snapshot in the Main Tab on the controller The same file convention is used but with the word Screenshot in the file name PAUSE STOF SNAPSHOT 16 2 888 744 2244 6 www sagescience com e support sagescience com Protocol Files The protocol file directory is accessed by pressing the GO TO PROTOCOLS button Protocol files have a ppprot extension and are stored in a Protocols directory with the name saved by the user Protocols may be copied to a flash drive for archiving or transfer to another Pippin Prep Cassette Files The log file directory is accessed by pressing the GO TO CASSETTE button Cassette files have a ppcass extension and contain the calibration parameters for the cassettes that are indicated in the file names Cassette files may be copied to a flash drive and transferred to another Pippin Prep Cassette files are not editable O Note Cassette calibration files may be updated during software upgrades Users should check release notes and save legacy cassette files if a methods have been developed with their use Managing Files The commands for managing files are found at the bottom of the File Manager Tab The functions are outlined below MEvY FLASH FOLDER UN MOUNT FLASH DRIVE Creates new Copies Copies folder on highlighted highlighted ad tidal sai A attached USB file s from file s from files flash dr
65. v Ta AA B TR AA N V l Pa ka I Pa c P 4 Am W Wi CX 4 tf T CO Pippin Prep Operations Manual software v 6 13 CD9 15 3 lt I 888 744 2244 6 www sagescience com 6 support sagescience com Signal Graph The Signal graph allows users to closely inspect the elution results of a Pippin run from a run log The colored horizontal traces display the actual optical signal results in mA The vertical red lines indicate called reference DNA marker peaks that are listed in the DNA marker times and values list There is an Analyze Peaks button to recall the markers The yellow vertical line indicates the estimated base pair position at a given run time The indicator may be moved see below along the x axis showing the relative base pair position If the perspective of the graph is changed see below the indicator will automatically default to the center of the x axis SIGNAL HEA i 0 82 DU 0 60 L 050 T 2 S 040 C1 UL 0 08 l l l l l l l ll 01 05 48 01 07 00 01 08 00 01 09 00 01 10 00 01 11 00 01 1200 01 13 02 p Time hh mm ss There are four icons in the upper right hand corner of the graph The functions are listed below Revert to default view Grab and move screen Move yellow Zoom base pair hold left indicator click for Options 15 4 888 744 2244 6 www sagescience com 6 support sagescience com Current Graph The Current graph allows users to closely inspec
66. w sagescience com 6 support sagescience com USB Ports 4 VGA monitor port Power Switch Figure 3 2 Rear Panel Ports Power Entry Port Mouse Monitor Cable Key board Power Cable Figure 3 3 Rear Panel Connections Pippin Prep Operations Manual software v 6 13 CD9 3 4 888 744 2244 6 www sagescience com e support sagescience com 4 sage science Figure 3 4 Pippin Prep front panel USB Port Indicator Light Blue when instrument is ready for use power is on and software is active Indicator Light Green when electrophoresis is underway protocol is running 3 5 888 744 2244 6 www sagescience com e support sagescience com Unpacking Gel Cassettes Gel cassettes are shipped in boxes in the following configurations Ensure boxes are in the upright position and confirm that following contents are present DNA size markers and loading solution should be stored at 4 C Storage of electrophoresis buffer at 4 C is optional The cassettes should be stored at room temperature e 10 cassettes with reagents to run 40 samples o 10 foil sealed gel cassettes store at R T o 1 reagent kit for 10 cassettes store at 4 C 40 ml of running buffer 440 ul DNA size markers external marker 500 ul loading solution external marker Or 440 ul marker loading solution mix internal standard o 1 package of adhesive tape for sealing elution wells Important DNA size markers and loading s
67. w sagescience com e support sagescience com 4 Adjust the run time value if necessary Run Time hh mm End Run when Elution is Completed Ei 5 Program the size selection protocol and assign the DNA reference marker lane There are four modes of programming on the Pippin Prep e Tight collects minimum allowable distribution range of DNA fragments using the median target base pair value e Range allows users to select the range to be collected using starting and ending base pair values e Time allows users to program extractions using the starting and ending elution time hr min sec values only a reference DNA marker is not used e Peak collects the next peak restriction fragment or PCR band after the set threshold base pair value has been reached Tight O Note Programming modes may be independently applied to individual sample lanes 6 Enter Sample ID or description optional 7 Press Save As button enter a name for the protocol 888 744 2244 6 www sagescience com 6 support sagescience com 11 3 Selecting a Cassette Definition Cassettes types are selected from the Cassette drop down menu Cassette files are presented in a list The appropriate cassette type to select is base on the cassette product that has purchased and in some instances the application to be used i e different electrophoresis protocols are used with the same cassette product Highlight the appropriate
68. ww sagescience com e support sagescience com 8 The LED photocurrents will automatically adjust to a factory setting and the Calibration Status field will contain the message Calibration OK LED Calibration Target ph mA Iteration LED Current counts Photocurrent mA EGE HEE HENE EE Calibration Status Calibration OK Prompt To restart calibration CALIBRATE To exit calibration window EXIT CALIBRATE EXIT Press EXIT to return to the Main Tab 888 744 2244 6 www sagescience com e support sagescience com 6 Preparing a Cassette 6 1 Visually Inspect the Cassette A Caution Some cassettes contain the DNA binding dye ethidium bromide Use appropriate protective equipment disposable gloves lab coat and lab glasses when handling cassettes 1 Remove the cassette from foil packaging Foil package is scored at one end to allow tearing 2 Inspect the levels of buffer in the buffer reservoirs Tip the cassette to consolidate any bubbles in the reservoirs then hold the cassette in a horizontal position and look at the reservoirs from the top and bottom edges Reservoirs should be nearly full of buffer and roughly equal in volume across the cassette If the buffer level in any reservoir appears less than 50 full compared with its neighbors the low reservoir should be refilled prior to running When refilling a visual check of buffer levels is sufficient a measured volume is not required
69. x Reference Lane APPLY REFERENCE TO ALL LANES USE INTERNAL STANDARDS 888 744 2244 6 www sagescience com e support sagescience com Protocol Parameters Detail The protocol parameters section is shown below Users will select a programming mode for each sample and a lane to designate as a reference Then the size selection values are entered either as base pair BP values or time values hr min sec in Time mode For Peak programming mode users enter a base pair value into the BP Thresh field the instrument will collect the next peak that it detects after that threshold has been reached Individual lanes may have different parameters and different modes Programming Ref Lane Base Pair Time Modes Indicators BP values values Tight Range Time eak Rer Lane T End TPause BP Thresh sample ID Template 11 2 Programming a New Protocol Quick Guide The following is a Summary of the steps required to program a protocol Detailed instructions are provided in the subsequent sections 1 Click the Protocol Editor tab along top of the screen 2 Select NEW to create a new protocol To edit an existing protocol press the LOAD button to select the protocol to be edited Protocol Changes NOT Applied amp SAVE AS 3 Select a Cassette Type from Cassette menu by clicking the folder icon Protocol fields cannot be edited unless a cassette type has been selected 888 744 2244 6 ww

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