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SDRB04-0

1. Mobility file 3100 POP6 BDTv1 Sequencing Analysis Software Vers 5 1 1 Run Module Run Temperature 55 C CTS2600 Leak Threshold 25 steps Current tolerance 100 uAmps Run current 100 uAmps Voltage tolerance 0 6 kVolts Pre Run Voltage 15 KVolts Pre Run Time 180 sec Injection Voltage 1 2 kVolts Injection Time 10 sec Run Voltage 15 kVolts Number of Steps 10 steps Voltage Step Interval 60 sec Data delay Time 240 sec Run Time 2600 sec Basecaller KB bcp Settings Sample Manager Basecaller KB bcp Settings Plate Record Dye Set E Dye set primer file KB_ 3100 POP6 BDTvl mob Mobility File 3100 POP6_BDTv1 mob Run Module CTS2600 6 2 Run Sequencing 1 Transfer your sequencing pipetting scheme into the Plate Record of the ABI PRISM 3100 Genetic Analyzer If the sequences should be later analyzed with the software Sequence Pilot JSI Medical Systems GmbH Kippenheim Germany see section 7 the sample naming conventions are Sample name_Amplification mix_ Sequencing primer Example Sample DRBO1 DRB E2F if amplification mix DRBO1 was used in the sequencing reaction with the DRB E2F sequencing primer 2 Place samples into the ABI PRISM 3100 Genetic Analyzer and run the instrument For details refer to the User Guides of ABI PRISM 3100 Genetic Analyzer and its softwares 7 Result evaluation For allele assignment the sequences are loaded i
2. Amplification of the HLA locus by PCR setup in pre PCR area thermal cycling in post PCR area Electrophoresis to check for positive amplifications gel control post PCR area Purification of the positive amplification products for sequencing post PCR area Sequencing reaction post PCR area Purification of the sequencing products post PCR area Separation of the sequencing products in the capillary sequencer post PCR area Sequence analysis and allele assignment with the Sequence Pilot HLA SBT software 5 1 Amplification Prepare PCR on ice gt Fill in your PCR protocol gt Label your PCR minitray gt Thaw PCR Buffer gt Pre mix 10 85 ul of PCR Buffer with 4 ul of 25 ng ul genomic DNA and 0 15 ul of Taq polymerase for each mix each PCR An excess volume to compensate loss during pipetting is recommended For example if you want to perform one CTS SEQUENCE HLA DRB1 test 12 mixes prepare a pre mix for 14 mixes 151 9 ul of PCR Buffer 56 ul of 25 ng ul DNA 2 1 ul of Taq gt Vortex the pre mix gt Pipette 15 ul of the pre mix into each well of the minitray 10 85 ul PCR Buffer gt Close the tubes and spin them down t4ul DNA 25 ng ul gt Put the minitray into the thermocycler and start the ampli 0 15 ul Taq Polymerase fication program CTS AMP see below 15 ul reaction volume Cave DNA resolved in buffers should always be diluted at least 1 1 with HPLC water prior to use in the
3. Cave ExoSAP IT is a viscous fluid vortex well before use and get rid of excessive enzyme hanging at the tip of your pipette Thermocycler program for purification with ExoSAP IT CTS PUR Step Temperature Time Numbers of cycles 1 37 C 15 min 1 2 80 C 15 min 1 3 4 C o0 Cave Do not forget to enter the reaction volume of 14 ul 5 4 Sequencing reaction General strategy For high resolution typing of HLA class II exon 2 must be completely sequenced If an allele is not separated by amplification we recommend to sequence in both directions forward and reverse to optimize base calling Mix DRB11 and DRB12 are especially designed to cover all HLA DRB1 allele groups in minimum by two amplification mixes on of the mixes DRB01 to DRB10 plus mix DRB11 or DRB12 This strategy is used to reduce the risk of an allelic drop out due to a failed amplification In case of homozygous results single allele in addition to the one positive mix of the mixes DRBO1 to DRB10 mix DRB11 or DRB12 if positive should be sequenced with DRB E2R or DRB E2F respectively Ifthe alleles are separated by amplification e i if two group specific mixes are positive it is sufficient to sequence the positive amplicons in only one direction we recommend to use the reverse primers Setting up a sequencing reaction gt Create a pipetting scheme determining which amplicon s and which sequencing primer s are pipetted into which p
4. Working Instruction CTS SEQUENCE HLA DRB1 For high resolution typing of HLA DRB1 Product No 237 Lot No SDRB04 0 For research use only University Clinic Heidelberg Department of Transplantation Immunology Im Neuenheimer Feld 305 69120 Heidelberg Germany Phone 49 6221 564013 Fax 49 6221 564200 www ctstransplant org Manual No 37 Page of 16 CTS SEQUENCE HLA DRB1 Revision April 03 2012 Lot No SDRB04 0 Content De TMOG Chi Ons cee cccseivesscss svccuscesvesuodesvecsdsesvetessesvesedeetessendost oseduetvosussesvcsuuesessuekestectidesves esse sveseesedsesesdeasetbdsudsetessaseee OD 2 Materials and Equipment s ccscsecoccsssescessssesenscsvasseseosadeoasessscesectesessenncssssesdecassn asec seseesecascosseenesassenseussvesoesesees O 2 1 Materials included in the CTS SEQUENCE HLA DRB1 Kit ccccccsccssseesceseceseeeseeeseeaceeaceeseeeseesseeesenenensees 3 2 2 Storage and CXPIT ALON sessen en eie ch detobaing EA ERE EEE EEEE EEE nate beset eshte 4 2 3 Materials and equipment not included cccscsccssesscesesssesecssesecuseescuseescesecseesecueeecuasesessecseesecaeesesnaseeeeaseaes 5 3 Preparation of buffers and agarose gel scccscscsssssssssssscssccssscsssssesecssecssecssesssssscsssssssssssssssssessscsssessoes 4 Isolation and concentration measurement Of DNA ssccccssssscssssscccsssscccsssscccssssccccssssccccssscccesssscccsssscccesss 7 5 TeSt Procedure sisscesaciiseacess
5. amplification buffers often contain PCR inhibitors e g EDTA Manual No 37 Page 7 of 16 CTS SEQUENCE HLA DRB1 Revision April 03 2012 Lot No SDRB04 0 Cave Do not use hot start polymerase e g AmpliTaq Gold Applied Biosystems or a proofreading polymerase Thermocycler program for amplification CTS AMP Step Temperature Time Numbers of cycles 1 95 C 2 min 1 95 C 15s 2 65 C 2 min X 95 C 15s 3 61 C 50s 22 72 C 1 min 30s 4 4 C 00 Cave Do not forget to enter the reaction volume of 15 ul 5 2 Gel control The amplification products are separated on a 2 agarose gel by electrophoresis This step is to check for success of the amplification step and to identify the amplification mix es which will be subjected to sequencing A Electrophoresis gt Pre pipette 5 ul of loading buffer for each amplification product into a PCR plate gt Add 5 ul of your amplification product Use filter tips to avoid contamination gt Load the gel with 10 ul of the amplification loading buffer mixture gt Ifyou use CTS electrophoresis chamber run the electrophoresis for 20 min at 170 Volts approx 0 4 V cm Cave Ethidium bromide is potentially carcinogenic Wear appropriate protection e g nitril gloves B Documentation and interpretation Place the gel on a UV light transilluminator 312 nm and take a polaroid picture for interpretation and documentation Wear UV protection goggles You can proceed wi
6. No 0030 077 040 Pipettes and filter tips for 0 5 10 ul volume Eppendorf Wessing Berzdorf Germany Cat No 0030 077 040 Multipette and combitips 0 1 0 2 0 5 1 0 2 5ml Not mandatory Eppendorf Wessing Berzdorf Germany Adhesive aluminium foils for 96 well PCR plate Kisker Steinfurt Germany Cat No GO71 Optical 96 well reaction plate and optical caps Applied Biosystems Darmstadt Germany Cat No N801 0560 N801 0535 Table 4 Pre PCR and post PCR area two sets are needed Reagents materials softwares Company Catalogue number HPLC water LiChrosolv water Merck Darmstadt Germany Cat No 1 15333 1000 Vortexer Reaction tubes 1 5 ml Eppendorf Wessing Berzdorf Germany Cat No 0030 120 086 Examination gloves Nitril gloves 3 Preparation of buffers and agarose gel 1x TAE electrophoresis buffer 49 volume parts of deionised water 1 volume part of 50x TAE electrophoresis buffer Ethanol 70 7 volume parts of absolute ethanol 3 volume parts of HPLC water 2 agarose gel If you use CTS electrophoresis chamber and CTS combs see www ctstransplant org for order information proceed as follows Manual No 37 Revision April 03 2012 Page 6 of 16 CTS SEQUENCE HLA DRB1 Lot No SDRB04 0 Add7 g of agarose and 7 ml of 50x TAE buffer to 350 ml of ddH 0 Boil to dissolve the agarose using a magnetic stirring hot plate o
7. eens 8 2 Sequencing Appendix Worksheet Amplification Protocol 15 Worksheet Pipetting Scheme cscscscccecccsccccscccsccscccscscscccsscssscsssscscssssssssers 16 Manual No 37 Page 2 of 16 CTS SEQUENCE HLA DRB1 Revision April 03 2012 Lot No SDRB04 0 The CTS SEQUENCE HLA B Kit is delivered at room temperature Immediately upon receipt store PCR Buffer amp sequencing primers at 20 C and PCR minitrays at 4 C 1 Introduction This working instruction describes the procedure for high resolution genotyping of the human leukocyte antigens HLA DRB1 with the CTS SEQUENCE HLA DRB1 Kit PCR sequencing based typing PCR SBT is an accurate and reliable method allowing high resolution of HLA alleles at least 4 digit level The strategy is based on two consecutive steps first group specific amplification of exon 2 of HLA DRB1 second the amplification products are sequenced in forward and reverse direction Matching for exon 2 antigen recognition site at allele level is considered relevant in hematopoietic stem cell transplantation The SEQUENCE HLA DRB Kit is validated and optimized with following reagents instruments softwares and methods GeneAmp PCR System 2700 Thermocycler Applied Biosystems Darmstadt Germany Amplification with the MBI Taq polymerase Fermentas St Leon Rot Germany Purification of amplification products with EXO SAP IT USB Staufen Germany Sequencing re
8. product s DNA contains PCR inhibitors Heparinized blood New extraction of DNA Thermocycler is defect Check cycler Some primary checks Did you follow the amplification protocol Did you vortex the solution well Was the correct cycler program used Was ethidium bromide included in the gel e g with the CTS Cycler Control Kit Incorrect thermocycler program Correct programm and repeat PCR Thermocycler program needs to be adapted Our method was optimized for the GeneAmp PCR System 2700 Thermocycler For other thermocyclers the cycling program may have to be adjusted and validated Taq Polymerase needs to be adapted Our method was optimized for the Taq DNA Polymerase purchased from Fermentas St Leon Rot Germany Cat No EP0401 EP0402 Repeat PCR with this polymerase 8 2 Sequencing Observation Possible Cause s Solution No signal No sample was in sequencing Repeat sequencing reaction reaction Not enough formamide or air bubble Pipette enough formamide and spin at the bottom of the well down well Weak signals Wrong injection time or injection Differences between capillary voltage sequencer can occur Adapt injection time or injection voltage to get fluorescent intensities between 400 and 9000 in raw data Not enough sequencing products after purification Cleaning up by ethanol precipitation requires very precise ethanol
9. B12 can only be sequenced in forward direction DRB E2F Table 1 Labeling of the sequencing primers HLA Locus Tube label Sequenced Not Applicable Direction of Exon for Mix sequencing DRB E2F 2 DRB11 forward Be DRB E2R 2 DRB12 reverse 2 2 Storage and expiration All kit components are labeled with storage condition and date of expiration Frequent thawing and freezing can reduce the quality of the reagents and should be avoided It is recommended to make aliquots of appropriate volumes and store them as indicated Manual No 37 Revision April 03 2012 Page 4 of 16 CTS SEQUENCE HLA DRB1 Lot No SDRB04 0 2 3 Materials and equipment not included Table 2 Pre PCR area Reagents materials softwares Company Catalogue number Taq DNA Polymerase 5 U l Fermentas St Leon Rot Germany Cat No EP0401 EP0402 Ultra Pure Agarose Inno Train Kronberg Taunus Germany Cat No GX04090 Ethidium bromide 10 mg ml Cave potentially carcinogenic Sigma Aldrich GmbH Steinheim Germany Cat No E1510 10ML Magnetic stirring hotplate or a microwave oven for gel preperation Pipettes and filter tips for 0 5 10 pl 10 200 ul and 200 1000 pl volumes Eppendorf Wessing Berzdorf Germany Sequence Pilot HLA SBT JSI Medical Systems GmbH Kippenheim Germany Photometer for spectral measurememnt of DNA concentration 50x TAE buffer Inno Train Kronberg T
10. BOS 850 _ 15 16 2 DRBO9 710 __ 10 2 DRB10 730 13 01 02 13 16 13 18 14 17 14 21 2 DRB11 850 01 03 04 08 11 12 13 14 15 16 2 DRB12 610 __ 07 09 10 2 Cave Mix DRB11 can only be sequenced with the sequencing primer DRB E2R and Mix DRB12 only with DRB E2F Comment Date Signature Operator Date Signature Reviewer Manual No 37 Page 15 of 16 CTS SEQUENCE HLA DRB1 Revision April 03 2012 Lot No SDRB04 0 Pipetting scheme Example Optical 96 well reaction plate 1 2 4 5 6 7 8 9 10 11 12 A Stan DRB02 _DRB E2F B Stan DRB02 _DRB E2R c Stan DRBO5S _DRB E2F D Stan DRBOS5S _DRB E2R E Stan DRB11 _DRB E2R F G H DNA sample ID Name e g Stan Amplification pattern of the B locus DRB0S positive DRB11 positive DRBO02 positive eee OOOO OOO0 Manual No 37 Revision April 03 2012 Position on plate Al A2 A3 A4 AS Page 16 of 16 Mix DRB02 was sequenced with the DRB E2F sequencing primer Mix DRB02 was sequenced with the DRB E2R sequencing primer Mix DRB05 was sequenced with the DRB E2F sequencing primer Mix DRB05 was sequenced with the DRB E2R sequencing primer Mix DRB11 was sequenced with the DRB E2R sequencing primer CTS SEQUENCE HLA DRB1 Lot No SDRB04 0
11. Twenty four 16 well PCR minitrays 12 wells contain dried primer mixes each tray for one HLA DRB1 typing Store at 4 C in pre PCR area 2 3 tubes of CTS SEQUENCE PCR Buffer 1400 ul Store at 20 C in pre PCR area 3 Sequencing primers 500 ul each DRB E2F DRB E2R Store at 20 C in post PCR area Manual No 37 Page 3 of 16 CTS SEQUENCE HLA DRB1 Revision April 03 2012 Lot No SDRB04 0 a PCR stripes and amplification mixes The amplification primers are prepipetted and dried in purple PCR stripes Note only 12 of the 16 cavities contain primers For quality reasons we recommend to use only the caps included in the package Figure 1 shows the positions of the PCR mixes on the stripe and the allele group s and the exons amplified by these mixes Mix Amplified Allels DRB12 07 09 10 x DRB11 01 03 04 08 11 12 13 14 15 16 x DRB10 43 01 02 13 16 13 18 14 17 14 21 DRBO9 10 x DRB08 15 16 DRB07 08 12 x DRBOS 11 13 14 except 14 03 14 06 13 15 14 20 DRB05 09 1 DRBO4 07 DRBO3 04 DRBO2 03 14 03 14 06 13 15 14 20 DRBO1 01 Black marker line Figure 1 Mix position on CTS SEQUENCE HLA DRBI tray The X marked wells contain no primer mixes o Sequencing primers The tubes containing the sequencing primers 500 ul have purple colored caps Mix DRB11 can only be sequenced in reverse direction DRB E2R and mix DR
12. action with BigDye terminator v1 1 Kits Applied Biosystems Darmstadt Germany Purification of the sequencing products using ethanol precipitation Resuspension of sequencing products with HiDi formamide Applied Biosystems Darmstadt Germany Separation of sequencing products with the ABI PRISM 3100 Genetic Analyzer Applied Biosystems Darmstadt Germany Sequence analysis and HLA allele assignment with Sequence Pilot HLA SBT JSI Medical Systems Kippenheim Germany Other reagents instruments etc may be used but should be validated by the user The CTS SEQUENCE kits have been validated to be performed with the GeneAmp PCR System 2700 thermocycler If other cyclers are used the ramp rate has to be set at 1 C sec According to EFI standards for histocompatibility testing Version 5 6 1 L3 2520 PCR SBT typing of HLA class II bases on amplification and sequencing primers which are located outside of exon 2 For many HLA class II variants only the sequence of the antigen recognition site exon 2 are reported Even though the PCR SBT HLA SEQUENCING Kits have been extensively tested and validated an allelic drop out of a rare or new allele due to mutations in the priming sites cannot be categorically ruled out 2 Materials and Equipment 2 1 Materials included in the CTS SEQUENCE HLA DRBI1 Kit The SEQUENCE HLA DRBI Kit provides reagents sufficient for twenty four HLA DRB1 high resolution typings and contains 1
13. aunus Germany Cat No GX12765 Analytical balance Manual No 37 Revision April 03 2012 Page 5 of 16 CTS SEQUENCE HLA DRB1 Lot No SDRB04 0 Table 3 Post PCR area Reagents materials softwares Company Catalogue number ExoSAP IT USB Staufen Germany Cat No 78202 BigDye Terminator Cycle Sequencing Kit v1 1 Sequencing buffer 5x included Applied Biosystems Darmstadt Germany Cat No 4336791 1x TAE electrophoresis buffer See section 3 below for instruction HiDi Formamide Applied Biosystems Darmstadt Germany Cat No 4311320 Loading buffer bromophenol blue Fermentas St Leon Rot Germany Sodium Acetat 3M pH 5 2 for precipitation Sigma Aldrich Germany Cat No S7899 Ethanol absolute GR for analysis Ethanol 70 10x EDTA running buffer for the sequencer Merck Darmstadt Germany Cat No 1 00983 1000 See section 3 below for instruction Applied Biosystems Darmstadt Germany Cat No 402824 1x EDTA running buffer for the sequencer Centrifuge for PCR plates GeneAmp PCR System 2700 thermocycler Applied Biosystems Darmstadt Germany Power supplier for electrophoresis Gel Documentation System Gel elektophoresis chamber Capillary sequencer ABI PRISM 3100 Genetic Analyzer Applied Biosystems Darmstadt Germany 8 channel pipette and filter tips 0 5 10 ul Eppendorf Wessing Berzdorf Germany Cat
14. cation of sequencing products did not work well leftover of dye Ethanol concentration during precipitation to high Very high randomly occurring peaks spikes Air bubbles or polymer crystals in capillaries Refill capillaries with new polymer Two different peaks run at nearly the same position in the electropherogram Secondary structures of sequencing products gel compression This phenomenon is sequence dependent and occurs only in one sequencing direction of a limited region Analyze this region with the sequencing primer for the other direction The sequences obtained with the forward primers tend to show gel compressions more often than reverse primers Manual No 37 Revision April 03 2012 Page 14 of 16 CTS SEQUENCE HLA DRB1 Lot No SDRB04 0 CTS SEQUENCE HLA DRB1 Amplification Protocol For Lot DRB04 0 DNA No Date Thermocycler Lot Volume PCR Buffer 10 85 ul TAQ 0 15 pl DNA 25ng pl 4ul The exact amount of Taq Polymerase needed may vary depending on brand and lot it should therefore be established through your own validation Photo Mix alice Peete Amplified Alleles pps DRBO1 840__ 01 2 DRBO2 990 03 14 03 14 06 13 15 14 20 2 DRBO3 760___ 04 2 DRB04 740 07 2 DRBO5 850 09 2 DRBOG 886 711 13 14 except 14 03 14 06 13 15 14 20 2 DRBO7 1030 08 12 2 DR
15. concentrations Ethanol concentration can vary when tubes are frequently opened Aliquot ethanol solutions for single use Not enough sequencing products were loaded Increase injection time or injection voltage Salt can reduce the amount of loaded sequencing products Reduce salt contamination during ethanol precipitation Signals are too strong Wrong injection time or injection voltage Differences between capillary sequencer can occur Adapt injection time or injection voltage to reach fluorescent intensities between 400 to 9000 in raw data High concentration of sequencing products Reduce the amount of PCR product used in the sequencing reaction The Manual No 37 Revision April 03 2012 Page 13 of 16 CTS SEQUENCE HLA DRB1 Lot No SDRB04 0 reduced amount should be substituted with HPLC water e g dilute amplicon with HPLC water Electropherogram has high background Purification of PCR amplification products did not work well primer contamination Repeat PCR and purification of amplification products Contamination with a second sequencing primer Avoid contamination during pipetting sequencing primers Double sequence which starts in the forward and reverse sequencing reaction at the same base in different directions Double sequence due to inserts or deletions within an HLA Bllele DyeBlobs Purifi
16. il flip the optical 96 well reaction plate and remove the supernatant Place the optical 96 well reaction plate upside down on paper towel into the centrifuge Spin the plate for a few seconds at 180 x g to dry Keep the plate in a dark place until all ethanol has evaporated 20 min In dried form the sequencing products are quite stable when kept in the dark 5 6 Sample preparation for sequencing runs Add 15ul of HiDi Formamide onto the dried sequencing products close the wells with caps and spin down Put the plate into a thermocycler and denature for 2 min at 95 C IMPORTANT Vapours at high temperatures Cool down the HiDi Formamide at 4 C before opening the caps 6 Start of a sequencing run on the sequencer 6 1 Instrument protocol for ABI Prism 3100 Genetic Analyzer Applied Biosystems Darmstadt Germany POP medium 3100 POP 6 Capillary 36 cm array Electrophoreses buffer 1x buffer with EDTA Instrument Protocol Type Regular Run Module CTS2600 Dye Set E Big DyeV1 Sequence File Format True Profile Ending Base At PCR Stop Do not assign N s to Basecalls Mixed Base Use Mixed Base Identification Call IUB if 2 highest Peak is 25 of the highest peak Clear Range Method Use quality values Remove bases from ends until viewer Manual No 37 Page 10 of 16 CTS SEQUENCE HLA DRB1 Revision April 03 2012 Lot No SDRB04 0 then 10 bases out of 20 have QVs less then 15
17. nto the Sequence Pilot HLA SBT Allele Identification Software JSI Medical Systems GmbH Kippenheim Germany This software shows the electropherograms and aligns them with HLA alleles as listed in the IMGT HLA Sequence Database http www ebi ac uk imgt hla Mismatches to the proposed HLA alleles if shown can be edited The sequencing results can be printed and archived For details see User Manual of the Sequence Pilot HLA SBT Allele Identification Software Add the sequencing primers with following names and parameters in the Seq Primer master file Manual No 37 Revision April 03 2012 Page 11 of 16 CTS SEQUENCE HLA DRB1 Lot No SDRB04 0 HLA DRB1 Name DRB E2F DRB E2R Gene DRB1 DRB1 Direction fwd rev SeqPrimer gene parts E2 E2 RFName DRB E2F DRB E2R Sorting 0 0 Adding the sequencing primer to the Seq Primer master file is not mandatory However by doing so one can avoid a situation in which a forward sequence of exon 3 is shown which has been sequenced by the forward sequencing primer of exon 2 such a sequence will have bad quality and can be omitted Manual No 37 Revision April 03 2012 Page 12 of 16 CTS SEQUENCE HLA DRB1 Lot No SDRB04 0 8 Troubleshooting 8 1 Amplification Observation Possible Cause s Solution Degraded DNA New extraction of DNA No weak or non specific DNA concentration to low New extraction of DNA PCR
18. osition s of the optical 96 well reaction plate An example of a pipetting scheme can be seen in the appendix gt Place an optical 96 well reaction plate on ice gt Mix one volume of BigDye terminators BDT with one volume of 5x BigDye sequencing buffer always prepare freshly Keep an excess volume to compensate loss during pipetting Pipette 2 ul of the mixture into the optical 96 well reaction plate Alternatively pipette 1 ul of BigDye terminators 1 ul of 5x BigDye sequencing buffer directly into the optical 96 well reaction plate Close the wells with caps and spin down gt Add 6 ul of sequencing primer gt Add 2 ul of purified amplification product DNA template 1 ul BDT gt Spin down close the plate with caps and place it into the thermocycler 1 ul 5x buffer gt Start the thermocycler program CTS SEQ 6 ul Primer 2 ul Template Cave Keep the BigDye terminators cool and minimize their exposure to light 10 ul Thermocycler program for sequencing reaction CTS SEQ Step Temperature Time Numbers of cycles 1 96 C 1 min 1 96 C 10s 60 C 2 min a 3 4 C 0 Cave Do not forget to enter the reaction volume of 10 ul Proceed with the purification of the sequencing products immediately when the sequencing reaction has finished Manual No 37 Page 9 of 16 CTS SEQUENCE HLA DRB1 Revision April 03 2012 Lot No SDRB04 0 5 5 Purification of the sequencing products Residual ddNTPs must be remo
19. r a microwave oven Cool down to 60 C add 17 ul of ethidium bromide 10 mg ml mix and pour the gel Allow the gel to set for 1 hour at room temperature Cave Ethidium bromide is potentially carcinogenic Wear appropriate protection e g nitril gloves Ona 20x25 cm gel you can place up to six CTS combs These combs have a tooth distance corresponding to that of the channels of a standard 8 channel pipette This allows the use of such a pipette for rapid loading of the samples onto the gel 4 Isolation and concentration measurement of DNA Genomic DNA can be isolated from all nucleated cells Starting material can be EDTA or citrate blood buffy coats cell suspensions etc Heparinized blood should not be used DNA can be isolated by the salting out method Miller SA et al Nucleic Acid Research 1999 or magnetic particle technology e g GenoM 6 Qiagen EZ1 robot Qiagen Vienna Austria Magnetic beads should be separated from the DNA e g by centrifugation It is likely that other commercial kits or automats for DNA isolation will also work but they should be validated by the users For optimal reaction adjust the DNA concentration to approximately 25 ng ul with HPLC water Cave Human material should always considered to be potentially infectious and be handled with care See your own standard laboratory safety guidelines 5 Test procedure High resolution HLA typing with the CTS SEQUENCE HLA DRB 1 Kit is performed in 7 steps
20. ssscenssacssscdasswassesontensdsccnseseasssoaessscsooaansdnacecadsesesonsacascb scesasossasosedsdenassonssseonsocansesbososoaes 7 5 1 Amplification eenean e e aad gaan buh eta EEEa A ea E E E E eda Seat cuba he Wade E ETE 7 3 2 6E0110 EEE E E ES 8 5 3 Purification of the amplification products sseseseseeseeeseeessesesesesressrsresrssesrresesressesresrssrerrnserreseseeeeseenes amp 5 4 Sequencing redchon merere naea Ea EE shea segs EEEE AEAEE ETES EAE EEE E E E REE 9 5 5 Purification of the sequencing PrOCUCHS ccccccesscesscesecesecnsecnseeseeeseeeseessceseceecesecesecsecaecuaecsaecaaeeseeeneees 10 5 6 Sample preparation for sequencing TUNS o cesccescesecsseesseeseeeseeeeeeseeesecesecesecsecuaecasecauecaeeeseeeaeeeeeeneseneeesees 10 6 Start of a sequencing run on the sequencer ssesessessescossesessoesesoossesoossesoesoesessossesosssesoesossossossessossesosssessesse LO 6 1 Instrument protocol for ABI Prism 3100 Genetic Analyzer sssoeseseeeseeeeseesrsreesrereerssrrerrsesrresesreeresee 10 0 2 R n SCQUENCING someren e e E E thE A seek E E EEE EEEE EEEE Suse OPETE EESE E EEE oE EENES eoe ll R 7 Result valuation E A A E T E A E E A E ORES ERR LL 8 T leshooti 13 rouUbleshootiNng o55 555cci5 cisco sek sucaccsadtenceceesticececestoasessducesaatencastsscusectscstegSeasdecesncuovsgsusseoesatinstscbecsoanecesvasacsecseceaes 6 1 Ampifican on aeoaea Sean begat hehe ta cages ok E E EE savas ca eases Sesh tade cu gens E AEA Hoan gies
21. th an amplification product if a band representing the specific amplicon is visible in the gel picture The length of the specific amplification products range from 610 to 1030 bp Cave Do not mistake primer dimers or primer clouds for specific amplification products Primer dimers are very small 15 50 bp Use a size marker if you are not confident 5 3 Purification of the amplification products Before an amplification product is subjected to sequencing it has to be purified e g with ExoSAP IT USB Staufen Germany ExoSAP IT contains an exonuclease digesting single stranded DNA e g primers and a phosphatase inactivating the nucleotides This enzymatic purification method is simple and appropriate to perform large scale testing A further advantage compared with other methods is that the enzymatic digest is performed in the same tube that will subsequently be used for the amplification step This avoids contaminations and a mix up of samples Add 4 ul of ExoSAP IT 2ul ExoSAP IT per 5ul PCR products to each well with a positive PCR reaction based on the gel control For large scale performances a Multipette can be used Close the reaction tubes avoid contaminations Spin down the ExoSAP IT in the reaction tubes Put the PCR reaction wells into the thermocycler and start the purification program CTS PUR see below VVV WV Manual No 37 Page 8 of 16 CTS SEQUENCE HLA DRB1 Revision April 03 2012 Lot No SDRB04 0
22. ved to avoid sequencing artifacts e g dye blobs This can be done e g by ethanol precipitation which is a cheap method and can be used for high throughput Pre mix 1 ul of 3 M Sodium Acetate pH 5 2 with 25 ul of absolute ethanol for each sequencing reaction to be purified An excess volume to compensate loss during pipetting is recommended Add 25 ul of the pre mix to each sequencing reaction Close the optical 96 well reaction plate with an adhesive aluminium foil and vortex well 30 sec Vortexing is crucial for a good precipitation Incubate the optical 96 well reaction plate at room temperature in a dark place for 15 min keep light exposure of ddNPTs low Centrifuge the optical 96 well reaction plate for 30 min at 2000 x g Proceed immediately with the next step If you can not proceed immediately centrifuge again for 3min at 2000 x g before the next step Remove the adhesive aluminium foil flip the optical 96 well reaction plate and remove the supernatant Place the optical 96 well reaction plate upside down on paper towel into the centrifuge Spin the plate for a few seconds at 180 x g to dry Add75 ul of 70 ethanol to the precipitated sequencing products and vortex briefly Centrifuge the optical 96 well reaction plate for 10 min at 2000 x g Proceed immediately with the next step If you can not proceed immediately centrifuge again for 3min at 2000 x g before the next step Remove the adhesive aluminium fo

SDRB04-0

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