Array View Troubleshooting
Contents
1. RawDataTroubleshooting Final Page 25 of 36 Applied Biosystems G or C Breakdown Formamide absorbs water in air undergoing hydrolysis to generate Formic Acid and Ammonia One of these is responsible for the degradation of the dyes To prevent the occurrence of dye breakdown it is recommended to run the samples immediately after resuspension or to store the samples seal the samples to prevent exposure to air G breakdown with BigDye Terminator V1 0 Shouldering of C peaks due to dye breakdown in BigDye Terminator V3 1 500 880 960 1040 1120 1200 GLO TGCOTTOTCOTCACTGTOTCCACTTCOTTGAACAAT fall a0 100 pul hd T Applied ES Biosystems ooting Final Applied Biosystems Late Start BA E eras Analysis 3 7 Lotes extreme abl Wi il RawDataTroubleshooting Final Module Page 28 of 36 Applied Biosystems Sample Transfer syringe and pump The red arrows identify areas of possible leaks From water reservoir to syringe From syringe to robot tip From water reservoir to syringe From syringe to robot tip Tips should be tightened until finger tight RawDataTroubleshooting Final Module Page 29 of 36 Applied Biosystems Array Diagram Load header Array base ks Capillaries a nd Sheath tip cower Loading end of Broken capillaries along the length of the array can be problematic if the array is not changed array Polymer will still flow through the capillary2. RawDataTroubleshooting Final Module Page of 36 Applied Biosystems Possible Causes Recommended Actions No Data Weak data Ft ay nod Int ngby we Hun Tim i lick to enlarge Weak data 58 56 58 06 58 56 70 06 70 56 71 06 Intensity vs Run Time mins Click to enlarge 71 56 For fragment analysis the capillary that is borrowed is further than 5 capillaries away If results are sporadic or random Sample Transfer problems Bubbles in the sample wells Bubbles in the sample transfer lines Clogged autoloader tips Sample Issues Module Background fluorescence Refer to the Array View Colors Troubleshooting Module to identify fluorescence Select a capillary that is within a 5 capillary distance If a capillary is unavailable run another spectral Refer to page 4 41 of the ABI PRISM 3700 DNA ANALYZER USER S MANUAL Reviewing and Overriding the Spectral Calibration Profiles Spin down samples before placing on the instrument Inspect the tubes for bubbles in the bottom of the well Verify that the fittings on the ports above the sample transfer syringes are finger tight Verify that the sample transfer syringes are tightened to the pump Finger tight should be sufficient Verify that the fluid line sinkers are at the bottom of the reservoir Verify water levels in the water reservoir Replenish if necessary Run the Change tips wizard This will prime the syringes and flush water through the tub
3. Select the run to display Run_my3700_1999 02 05_13 OF Cancel 3 From the drop down list box select the run that you want to display Click OK This opens the Retrieving block box PU Retreiving block 0 from Run my3 Fatreiving block 0 from Run my3 00 194 4 When the retrieving window disappears go the to run status tab and select the array view RawDataTroubleshooting Final Module Page 11 of 36 Applied Biosystems ae LI LI Lan 1700 Cats kalii He EESTI Yarn 7 Uhl g Fie Edl vies dnzbrament Help Dele Aegidii ad ER l l Piste Setup Run Setup Pn Status Fun Log Emmius rer view En anry view CCD wiw laba rate oar Ti rren rri roth 4i zu 1200 e 31 Bret Electropherogram i display ert Capilar Number 43 Dtconrecting from Aubo Extraction Server Use the capillary selector slider to view individual capillaries in the electropherogram view on the left Module Smp Stmopeed because of error LL EE Data is stored in blocks Use the right scroll bar to scroll through the blocks of data RawDataTroubleshooting Final Page 12 of 36 Applied Biosystems What is the electropherogram display An electropherogram is a graph of relative dye concentration against time plotted for each dye The electropherogram display is plotted using Intensity vs Time The plot also appears in the capillary view tab but has been rotated 90 degrees this
4. RawDataTroubleshooting Final Module Page 7 of 36 Applied Biosystems Sample issues Run the long read standard to verify the instrument is running properly if so please contact technical support for sample related issues Failing samples with signal Broken capillaries Inspect the capillary array for broken capillaries replace defective lt 50 RFU arrays Improperly installed array load Confirm that the capillary array load header is properly seated and header that all capillaries are in their proper wells Non optimal cuvette temperature Choose the cuvette temperature with the most uniform signal strengths across the array for all subsequent run If problem persists please contact technical support Contacting Technical Support By phone 1 800 831 6844 option 5 By email ABTechnicalSupport appliedbiosystems com RawDataTroubleshooting Final Module Page 8 of 36 Applied Biosystems Restarting the 3700 DNA Analyzer IMPORTANT To prevent firmware and software memory problems we recommend that you restart the instrument and the software once a week To shut down and restart the instrument Step 1 The instrument should not be running or extracting data Step 2 Close the 3700 Data Collection software by selecting Shutdown from the File menu Note You cannot use the Close button to exit the software Step 3 Close the OrbixWeb Daemon software by right clicking on its button in the taskbar and selecting Close from the pop up m
5. 36 Applied Biosystems After selecting run method the steps of the service module will be listed d Run Service Modules INSTRUM 1 AMMAN J RRSP You must choose a file to run You selected module file CuvetteFlush maod Begin Cuvette Flush Module Assumes cuvette and lines are filled with polymer The module selected should be listed Initializing sensors Flushing tubing to cuvette with POP Filling load trough weith water Flushing tubing to cuvette with POP Flush twice wih polymer pump while the sheath pump is flushing aiting for sheath pump Flush polymer through cuvette out valve 11 waiting for sheath pump Flush 2 5 ml out valve 11 with sheath pump Flush 4ml polymer out valve 11 with polymer pump while sheath pump iz running il Finished 3ml aiting for sheath pump H Allow pressure to return to zero Flushing bubbles from cuvette Pressurize to 60 psi for 1 minute Opening cuvette to purge bubbles Flush polymer through cuvette with polymer pump out valve 11 then out valve 12 Repeat flushing bubbles from cuvette Pressurize to 60 psi for 1 minute Flush polymer through cuvette with polymer pump out valve 11 then out valve 12 Third time flushing bubbles from cuvette BI Fressurize to 60 psi for 1 minute Flush polymer through cuvette with polymer pump out valve 11 then out valve 12 Flush 1 5ml polymer out valve 12 with sheath pump Flush 2ml polymer out
6. If results are have a pattern Refer to the Missing Lanes Module to for more details Spikes or Bright Events A leak in the system allowing Inspect polymer lines for any source of bubbles or particles correct particulates to enter the system any leaks Replace the inline filter Replace polymer with a fresh bottle Perform a regenerate array Bubbles in the cuvette or the array Run the Cuvette Flush module Refer to the Checking for Bubbles Module and the Checking for Leaks Module if the bubbles in the cuvette persist RawDataTroubleshooting Final Module Page 3 of 36 pplied Biosystems Observations Possible Causes Recommended Actions Use an Uninterruptible Power Supply UPS Capillary specific Run a regenerate array to flush out of the capillary refer to the Click to enlarge Change Array wizard and select to clean the array This may not always be successful depending on the contaminant If the spike does not go away one can block the capillary within the software refer to page 4 12 of the ABI PRISM 3700 DNA ANALYZER ee ee ge USER S MANUAL Over tightening the thumbscrews Remove the array and re install such that the thumbscrews are on the detection end of the array tightened until finger tight Check the CCD View to confirm the The laser beam may simply be too presence of horizontal rows of bright spots near the bottom of the close to the capillaries resulting in CCD display window typically referred to as p
7. causing buildup of polymer in the area and affecting other capillaries Detection end of array Check the area for any polymer leaks if present remove the array and clean the area of polymer Clean with a lint free tissue moistened with DI water See below to check the o ring Thumbscrews These should be tightened until finger tight RawDataTroubleshooting Final 1VIVUUILY Page 30 of 36 Applied Biosystems Load Bar EIBIM EH PIELE ERS PI ies LI LE ET a DIM MU a delete SEUL RUES M OE Sete ali Verify that the capillaries are sitting in the wells of the load bar and that they are covered with water or Load bar buffer Inline Filter Dried polymer will appear in these areas Inline filter zx RawDataTroubleshooting Final Module Page 31 of 36 Applied Biosystems Reservoirs and Fluid Line Sinkers The Buffer and Water reservoirs should have enough fluids for the scheduled runs It is recommended to check the reservoirs daily Also verify that the fluid line sinkers are at the bottom of the reservoirs and that the lines are submerged under the fluid Fluid line sinkers Buffer Water Running the wizards Step 1 Select Instrument gt Select Wizards gt Select the appropriate wizard Instrument EXIT Cir SR Ce Stap iri Wizards Change Array Utilities F change Syringe Manual contral Change Polymer Change Tips Longterm RawDataTroubleshooting Final M
8. is from a long read standard zu D 400 31 38 81 88 a238 52 55 53 35 53 55 54 38 Intensity vs Run Time ming RawDataTroubleshooting Final Module Page 14 of 36 Dye sets Filter Set TO O I M G5 E5 Dyes or kit BigDye Terminator V1 1 BigDye Terminator v3 1 6 FAM HEX NED and ROX 6 FAM VIC NED and ROX 5 FAM JOE NED and ROX 6 FAM VIC NED PET and LIZ dR110 dR6G dTAMRA dROX and LIZ Module Applied Biosystems RawDataTroubleshooting Final Page 15 of 36 Two Lines per peak Changing the preferences Applied Biosystems 1 Inthe Data Collection V2 0 Software select the Edit menu gt select Preferences gt select Run Status tab in the Setting Preferences window 2 Inthe Data Acquisition Option deselect the Display Confidence Bands Setting Preferences Users Instrument Plate Setup Run Setup Run Status Data Analysis Data Extraction Status Options Refresh Interval Range 0 30 Seconds F Seconds Record Interval Range 0 300 Seconds E Seconds Data Acquistion Options qu Display Confidence Bands OK Cancel Apply Module The Display Confidence Bands allows for the evaluation of the quality of the spectral The confidence bands provide a measure of the statistical confidence with which the electropherogram data is known A small confidence interval indicates a good spectral calibration for that capillary A large confidence i
9. valve 12 with polymer pump while sheath pump iz running Filing load trough with water E Run Service Modubrs RawDataTroubleshooting Final Select Run Method Module Page 35 of 36 Applied BS Biosystems oe Information e When this window appears the module has completed if there is an error during the module please contact technical support RawDataTroubleshooting Final Module Page 36 of 36
10. 5561 ee K ty omina Mj fe Edt gape Menage Window Hep ale aeu 128 55 17 GS 54 57 a mE N il ull Mi Anl n i al hac Uu v PI JL m M ka et Ld fi J iy T Loss Le E n 64 27 li ll 53 97 dH ml NULL a oe WA Y NS T m oe 53 57 a IEL 53 37 EL 63 07 md l T l I AUD I m M f lu 1 A RUM LL nut wu y JL I ALL p AM a JJ AL L Aa 562 77 ANI j Lal il B2 47 T um Li Nn bl LA ne AN I L J JJ i SET aT Capillary Number 20 The intensity of the spike is near 15 000 The sample is a long read standard which has an intensity of 800 1200 but due to the intensity of the spike the sample appears to be weak It is important to note the scale of the intensity vs time graph Also note that the spike generates a large negative peak due to the intensity of the spike the spectral can not adequately compensate for the amount of signal observed It is common for spikes to also exhibit negative peaks RawDataTroubleshooting Final Module Page 20 of 36 Applied Biosystems The array view sub page A bright event occurring in one capillary can affect neighboring capillaries The intensity of the spike will be much less than the capillary of origin RawDataTroubleshooting Final Module Page 21 of 36 pp
11. Applied Biosystems ABI PRISM 3700 Instrument Hardware Raw Data Troubleshooting Module This module is intended to troubleshoot raw data that are displaying anomalies in the electropherogram display in Data Collection Software V2 0 or the raw data profile in the analysis software BEFORE PERFORMING ANY TROUBLESHOOTING WORK ON YOUR 3700 INSTRUMENT PLEASE READ THE ABI PRISM 3 00 DNA ANALYZER USER S MANUAL FOR SAFETY AND WARRANTY INFORMATION AND FURTHER DETAILS ON USE OF THE SYSTEM Please contact technical support if you have any questions Text in blue indicates a link to another portion of the document Possible Causes Recommended Actions Two Lines per peak The preferences to display the Change the preferences to deselect the display confidence bands confidence bands were turned on Click to enlarge or view more examples Pull Up and or Pull Down Peaks Incorrect spectral is selected Rerun the samples verifying that the correct spectral dye set for your chemistry is being used Optics have been realigned or Rerun the spectral calibration replaced A spectral will need to be run for the following changes Changes to the optics Different polymer type is being A different polymer type is being used used Switched to POPS or POP6 New dyes are being used on the system For example in sequencing moving from BigDye V1 1 to BigDye V3 1 or in fragment analysis changing DS 30 to DS 33 0 87 79 88 29 88 79 Int Click to enlarge
12. ation system produces water purified to 18 Mega Ohms resistance Use a different water source if available Buffer or polymer levels are Prepare fresh 1X running buffer and fill the reservoir Place a new depleted bottle of polymer on the system Bubbles in the cuvette or capillaries Check for bubbles within the CCD view refer to Checking the CCD are blocking current flow window Module Run the cuvetteflush mod service module Please contact technical support for sample related issues Some samples have data start Overloading of sample due to the Use HiDi Formamide or increase the resuspension volume Contact points that are later than most of the high efficiency of injection from technical support for more information on decreasing sample signal samples of the run water intensity Data in the late starting lanes suffers early loss of resolution Run the Long Read Standard to verify instrument is running properly es LB eus See pem m at m D Old or expired reagents which can Replace the buffer or polymer with fresh supply Verify that the affect current reagents have not expired and have been on the instrument less Mis than 7 days Mis mal O lick to enlarge Low read length on one side Failing samples with signal gt Clumped capillaries Inspect the array for any capillaries that appear to be clumped 50 RFU together Carefully clean and separate clumped capillaries in place with damp tissue
13. d bleach in water followed by a thorough rinsing with deionized water Microbial contamination of water and or buffer reservoirs Click to enlarge Elevated Separation of the Baseline Background Fluorescence Refer to the Array View Troubleshooting Module for more information RawDataTroubleshooting Final Module Page 5 of 36 LN T F Dag rae a refit wi Run Tima rossi Click to enlarge G or C dye breakdown Click to enlarge Peaks appear as doublets or have bulges or small adjacent extra peaks ppuITIP Click to enlarge Gradual loss of resolution Contaminant present but electrophoreses out before data is collected Chemical breakdown of BigDye Terminator G dye or C dye Excessive sample has been electrokinetically injected into capillary Changes in the state of the capillary walls can lead to degradation of performance Module Applied Biosystems Possible Causes Recommended Actions There may be a contaminant in the system which appears early in the run before the samples The software incorporates this contaminant as part of the background level However the contaminant then electrophoreses out of the system before the samples appear therefore the amount of background changes It may not affect the analyzed data depending on the fluorescent intensity of the contaminant Refer to the Instrument Maintenance Module Minimize exposure to air for samples re sus
14. enu If you get a run time message click OK to close the message IMPORTANT Do not shut down the OrbixWeb Daemon until after you have shut down the Data Collection program Step 4 a Turn off the instrument using the On Off button The following diagram shows the left front grill TT TES Green ISRBERELERUI Yellow Status lights Red On Off button b Wait 30 seconds c Turn on the instrument Step 5 When the green status light is steady wait 1 minute Step 6 Restart the 3700 Data Collection software RawDataTroubleshooting Final Module Page 9 of 36 Applied Biosystems How is the array view accessed in Data Collection V2 0 During a run Note Always exit the Array View when you are finished viewing Do not leave the window open for extended periods during a run as this may cause unrecoverable screen update problems 1 Select the Run Status tab gt select Array View Below is a normal view of a run Array View Sub Page i 3700 Data Collection Software Version 1 1 Fie Edt View Instrumeot Help Deda Accusition Capillary status bar Electropherogram Capillary display display Capil sr Number 62 Capillary selector slider RawDataTroubleshooting Final Module Page 10 of 36 Applied Biosystems After a run Accesses information within the database 1 Select the Data Acquisition menu and select Display Reduced Data for Run 2 This opens the Select the run to display dialog box
15. icket fence Refer to direct illlumination of the tips the Checking CCD Module for more details on checking the CCD view Capillary Hurnber 20 Click to enlarge Bubbles migrating through the Run the Cuvette Flush module Refer to the Checking for Bubbles capillary degrade resolution and Module and the Checking for Leaks Module if the bubbles in the generate a bright signal upon cuvette persist emerging from the capillary tip within the cuvette Click to enlarge Data preceding the spike may have lowered resolution RawDataTroubleshooting Final Module Page 4 of 36 Applied Biosystems Possible Causes Recommended Actions opikes underneath the baseline opatial calibration may have shifted Run another spatial calibration and run Long Read Standard sample Fe Click to enlarge Jagged Peaks re Fluid other than polymer is mixing Avoid mixing polymer from different bottles Run the change polymer in the cuvette resulting in refractive wizard for a different lot if the lot numbers are different index changes and deflection of the laser beam Inspect sheath flow syringe for signs of fluid mixing during filling Install a new inline polymer filter and run the Cuvette Flush module to thoroughly flush cuvette Click to enlarge oymptoms are more evident for longer fragments Dark lines seen across array view Rinse the water and buffer reservoirs with either 1 hot water or 2 a 1 3 dilution of househol
16. ing going to the loading tips During the wizard monitor both sample transfer syringes for bubbles If bubbles are present in the syringe but not the tubing from the water reservoir change the syringe Replace the autoloader tips Refer to the change tips wizard for instructions Run the Long Read standard to determine if the issues sample related and then contact Technical Support RawDataTroubleshooting Final Page 2 of 36 Applied Biosystems Possible Causes Recommended Actions If results are capillary specific Capillaries are clogged Run a regenerate array to flush out of the capillary refer to the Change Array wizard and select to clean the array This may not always be successful If the capillary continues to fail one can block the capillary within the software refer to page 4 12 of the ABI PRISM 3700 DNA ANALYZER USER S MANUAL manually overriding the capillary state Or replace the array Capillaries are broken Replace the array Capillaries are not seated properly Verify that the array loading end header is seated correctly not under tension and the capillary tips are positioned in their injection wells Inaccurate spatial calibration Run another spatial calibration If results are across the entire array Sample Transfer pumps may not Refer to the Verifying Syringe Pumps Module for more details be functioning Electrophoresis issues Refer to the Electrophoresis Module for more details
17. ks Intensity ws Run Time mins If there are consistent patterns in the colors that are pulled up or pulled down for example green always below blue this can be attributed to an improper spectral being applied to the sample The function of the spectral is to subtract any overlap between the dyes If the pull up and pull down does not exhibit a consistent pattern then it may be sample related RawDataTroubleshooting Final Module Page 18 of 36 Applied Biosystems Weak Baseline or Flat Baseline Sequencing standards typically give peaks above 800 Examples No sample injection Weak injection The scale is only 100 and the baseline remains near 0 The scale is 0 200 but the peak heights are at 125 and below 100 200 100 100 BS 56 69 06 69 56 70 06 70 56 71 06 71 55 6S 56 69 06 Intensity ws Run Time mins 69 56 70 06 70 56 741 06 747 56 Intensity vs Run Time mins RawDataTroubleshooting Final Module Page 19 of 36 lied Biosystems Spikes or Bright Events The presence of contaminants or bubbles in the system can cause a brief intense signal when hit by the laser beam Spikes in the raw data can also be due to voltage spikes from either 1 other instruments on a non dedicated line or 2 from a non UPS surge protected circuit The spike can be in all four colors or a specific color depending on the cause and contaminant Intensity ws Time mins aji Segeencing Analia ih nzurmzigz ca DOT FV 12807 CT _ 20
18. lied Biosystems Anomalous Peaks Trace coco acadAcaraa crac ac craaTT ara TST OT T eT TO Tit TACT QUTO 5 STO C TOC TOCTUC TT OT OT OAC TOT CT CCC TTC OT Ta oc ET QC OC 6G 1G zu ja LLI r LE FL an E pug Iig TA ouh dt en dat a AAAA a Annaa aal f ruf adhah Nul VON ule Mini VAL RARI THUS H Jl Jes rosTOOCTTOTTTTOD OT OLOOTO TOO AQ amp amp p TaGOC coc dl orice Ne oso cuc OT OTT Que OC OT TOO TTT an XTTO DDR TIC TAT QT CN CC Teen ToT Q Ts acaafiraa sa a AAT Ta agca srcTTrasaa AT Te ee TT TOTO TO OT ess 420 Oooo ooo cos M re arc ooo Bib rr TT c HT FIT CH ETC ETT dr E pee CPE onam an ni es ac Abe coccooTTTTTAaZgau TTT amp aaanaconcoTTTTTTTTT S 5c acc ocMNaaNINTTTTTTTTTTTC Ma ee Tr TT amp a Sk D C it eaa id fom Bia EEL RawDataTroubleshooting Final Module Page 22 of 36 BS Bee ems Spikes underneath the baseline Sequencing Amalya 1 5 tert AMS 118 sample abi qid l CVO ww RawDataTroubleshooting Final Module Page 23 of 36 Applied Biosystems Jagged Peaks Striations F 1700 Dala Collection Software Version 1 1bhlci Fir ek ew mnt He etn Acquistion 5d miel Pinte Setup Mur Setup Pin Status in Los LE ME 35 SE BOT WAS DDA bad ab M pue Edt Same anao ire Help Jal xj 4 P _ W m TH zn TENT B Staha Amar views cepere wiew n emm wm Time mins nc en re dd oiu mus oe ji iI n
19. n P h IN Capillary Number amp Ny FX M P i ut i fa A ni I i I J yA a OT on mma Lt L d j4 7 At fi A stop TUCO OR NO CIEL E rem hi xs jm Elevated Separation of the Baseline 200 N 100 100 143 3 143 9 150 5 Intensity vs Run Time mins RawDataTroubleshooting Final IVIOQUIE 151 1 151 7 Page 24 of 36 Picket Fence Je ES Gress uerus sk zi ur i z bin ipi DEZ dam 7 TX Een I cul F mt dil zm He 13 IH MEL EMI Er E cm n IF Wini YA TIH Hie rig ajari ME WC i lip P i JH PTE ee waw es perme 5 oe LE l a SR acc ans aaa a n CUS 5 IX gm x cx X XE 4 de wi t5 gd rb och ili NE EE ix aJ Dn xa ia 5 Ili an LI 1 LI LI LI LI LI LI LI LI ET c2 EX i6 XT cO JC Ch Jc Kai ro PMC gne quu o BT X DEN Pam rro gura en HR E Fu To l Module lied Biosystems The picket fence is seen only on the right side and is minimal No action will need to be performed The picket fence observed to the left appears across the entire array If data is affected it may be necessary to adjust the array at the detection end tightening the thumbscrews until finger tight If this does not resolve the issue replace the array
20. nterval indicates a potential problem with the spectral data For more details on this please refer to the ABI PRISM 3700 DNA ANALYZER USER S MANUAL RawDataTroubleshooting Final Page 16 of 36 Examples of two lines per peak Example 1 Intensity ws Time mins 2d 200 400 B S9438 24 08 3 78 53 45 53 15 2 58 2259 oe 31 98 31 65 mx Capillary Number 1 Applied Biosystems Example 2 Example 3 Le Intensity ws Time mins 200 S00 1600 24 sq 2438 BD 34 08 400 200 2378 Y 23 48 200 aT 79 58 29 99 79 20 20 2 70 Di 23 15 Intensity ws Run Time mins 52 55 22 55 ae ts 21 95 21 65 21 329 Capillary Number 96 Example 1 shows a good quality spectral Example 2 shows the optimal spectral quality as the peaks appear to be a single peak Note that this sample is the Long Read Standard run immediately after the spectral calibration and that the spectral file is not borrowed from another capillary Example 3 shows a questionable spectral applied to a long read standard Note the separation of the peaks and also the pull up and pull down of the dyes under the main peak The spectral should be re run Li a Module Page 17 of 36 Applied Biosystems Pull Up and or Pull Down Peaks This exhibits both pull up and pull down in the baseline Pull down in green can be seen under the yellow and blue peaks ial inns M april palude M nice Pull up in red can be seen under the yellow pea
21. odule Page 32 of 36 AS Running service modules within Data Collection V2 0 Hun Service Modules Step 1 Go to the Instrument menu gt Select Utilities gt Select Run Service Module gt Step 2 Click the Select Module button es 3700 Data Collection Software Version 2 0 a b INT LT E L pa Ins E Poi Sio TEUA E E Eni Module Applied Biosystems RawDataTroubleshooting Final Page 33 of 36 Service Module Location e Service Modules a3 Us IL ee UracleDB D 2 AppliedBio 23 Support Files _ Data Collection Support m EService Modules ga Programs E eS IR Look in Files of type YAN Files d Cancel Ki E3 SE El AutoPlateDeck Calibration mod PopT of aterE change E BubbleRemove mad E FostB atchintemalu dU P One Level El CuvetteF lush mad E FreB atchlntemalll se nly mod a Fipet T est384 mad E 5 RSuringePrime mad E FipetTest3b5 mad Ej Ww aterF lush mod E FlenumPurge mad E W aterT aPapEschange mad Service Module Location E ervice Modules Look ir Files of type MAI Files Cancel Module Applied Biosystems The service module utility should automatically search the Service Modules Folder if not the folder can be found in the D AppliedBio Support Files Data Collection Support Files Service Modules Double click on the module in this example CuvetteFlush mod RawDataTroubleshooting Final Page 34 of
22. pended in HiDi Formamide by covering the samples with the recommended foil or a heat seal film and use the robot piercing Formamide absorbs water in air undergoing hydrolysis to generate Formic Acid and Ammonia One of these is partially responsible for the degradation of the extension fragments The recommended injection solution is HiDi Formamide because it is less prone to evaporation provides denaturing capabilities and yields the most consistent results However water can also be used for resuspension of samples but due to extremely efficient injection it generates the most signal but can also lead to excessive sample being injected If water is used it may be necessary to increase the resuspension volume Run the Long Read Standard sample using HiDi Formamide to eliminate instrument issues Run a regenerate array to flush out the capillary every 80 100 runs if maintained properly the array should provide 300 runs If the regenerate array procedure does not work replace the capillary array RawDataTroubleshooting Final Page 6 of 36 Applied Biosystems Possible Causes Recommended Actions Old or expired reagents Replace the buffer or polymer with fresh supply Verify that the reagents have not expired and have been on the instrument less than 7 days Poor quality water in buffer solution Remake 1X running buffer with fresh distilled deionized water or incorrectly made buffer Filtered water is adequate if the filtr
23. shows capillary number vs run time This view provides a quick view of capillary specific issues To access this view select the Run Status tab gt select the Capillary View tab Fa 3700 Data Collection Software Version 2 0 File Edt View Instrument Help Data Acquisition zs le Plate Setup Run Setup Run Status Run Log Status andi View Capillary View CCD View 2000 1600 1200 400 g1 38 81 88 52 35 52 55 23 38 53 55 54 38 Intensity vs Run Time mins RawDataTroubleshooting Final Module Page 13 of 36 Applied Biosystems Normal Data Below is how normal raw data should look and a general overview of what the plot displays Example 1 Data from an empty well 100 Notice the baselines for all colors are close together in L the blank injection Samples typically will not show such a baseline 10 129 47 130 07 130 67 131 27 131 57 132 47 Intensity ws Run Time mine The Y axis represents the intensity This scale will vary depending on sample and may be misleading if any offscale data exists as the sample may appear weak so it is important to note the scale of good data In this example the scale is 0 to 100 e The X axis represents the time in minutes Note that the peak morphology may differ depending on the part of the run that is being viewed Example 2 Long read standard results 2000 bao 1200 S00 For high quality samples the baseline can range from 0 200 This sample
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