SDQB04-0
Contents
1. Working Instruction CTS SEQUENCE HLA DQB1 For high resolution typing of HLA DQB1 Product No 338 Lot No SDQB04 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 38 Page of 16 CTS SEQUENCE HLA DQB1 Revision March 8 2012 Lot No SDQB04 0 Content De TMOG Ons seeseccasvesecss vccucsesvesesdesvessdsutvesosdesvciasseteoseudecesseteesessuacess suuidesvstuadsitesessesvetusdessecvdsesees cddustesesesdeosinsed D 2 Materials and Equipment cs cciscecocsssssscesssvessssssvasssssosesecseeasossovvesascassessesectocssseavecsssessesassssescossssesesseneseeses O 2 1 Materials included in the CTS SEQUENCE HLA DQBI Kit cscccsccssseesceeseeeseeeecesecuseensecasecaeeeaeenseennees 3 2 2 Storage and EXPT ALON senenin Noes ei bases EE EE Sede vats EEE Was soe EE E EEEE EE EAEE a 5 2 3 Materials and equipment not included sccscesccscessceseenseseeseeecscesscueeescuseeseesecseesecueeeeesaeeceaeeasesesaeeseenaes 6 3 Preparation of buffers and agarose gel sscssssssssscessesssessesecscecscecssccsscssscssscsssscsssssssssecssesssessccssecsseses 7 4 Isolation and concentration measurement Of DNA cssscccssssscccssssccecssscccsssscccsssseccccssssccsssccccssseccccsssees 7 S gt TeSt Procedure visscscsssstececssseccassessssck2. 510 bp 02 2 DQB02 500 bp 03 01 01G 03 04 2 DQB03 480 bp 03 04 03 01 01G 03 04 not amplified 2 DQB04 440 bp 05 2 DQB05 480 bp 06 06 01 not amplified 2 DQB06 530 bp 06 01 2 DQB07 660 bp All HLA DQB1 Alleles 2 DQB08 340 bp All HLA DQB1 Alleles 3 Cave Use DQB E2F and DQB E2R for sequencing of Mix DQB01 07 and DQB E3F and DQB E3R for sequencing of Mix DQB 08 Comment Date Signature Operator Date Signature Reviewer Manual No 38 Revision March 8 2012 Page 15 of 16 CTS SEQUENCE HLA DQB1 Lot No SDQB04 0 Pipetting scheme Example Optical 96 well reaction plate 1 2 3 4 5 6 7 8 9 10 11 12 A Stan DQB01 _DQB E2R B Stan DQB02 _DQB E2R c Stan DQB07 _DQB E2F D Stan DQB08 _DQB E3F E Stan DQB08 _DQB E3R F G H DNA sample ID Name e g Stan Amplification pattern of the DQB1 locus amp DQB08 positive Q DQB07 positive OOOO amp DQBO02 positive DQB0I positive Manual No 38 Revision March 8 2012 Position on plate Al A2 A3 A4 AS Page 16 of 16 Mix DQBO1 was sequenced with the DQB E2R sequencing primer Mix DQBO02 was sequenced with the DQB E2R sequencing primer Mix DQBO07 was sequenced with the DQB E2F sequencing primer Mix DQBO08 was sequenced with the DQB E3F sequencing primer Mix DQBO08 was sequenced with the DQB E3R sequencing primer CTS SEQUENCE HLA DQB1 L
3. 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 DQBI Kit is performed in 7 steps 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 Manual No 38 Page 7 of 16 CTS SEQUENCE HLA DQB1 Revision March 8 2012 Lot No SDQB04 0 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 DQBI test one stripe 8 mixes prepare a pre mix for 10 mixes 108 5 ul of PCR Buffer 40 ul of 25 ng ul DNA 1 5 ul of Taq gt Vortex the pre mix gt Pipette 15
4. 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 Vv 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 co 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 e i if only one of the group specific mixes DQBO1 to DQBO06 the locus specific mixes DQB07 and DQBO08 are positive we recommend to sequence the group specific mix in both directions forward and reverse to optimize base calling Incase of homozygouse results single allele mix DQB07 should be sequenced in both directions DQB E2F and DQB E2R to reduce the risk of allelic drop out 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 Sequencing of exo
5. 00 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 reaction 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 c
6. E1510 10ML Magnetic stirring hotplate or a microwave oven for gel preperation Pipettes and filter tips for 0 5 10 wl 10 200 pl and 200 1000 ul 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 Taunus Germany Cat No GX12765 Analytical balance 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 7899 Ethanol absolute GR for analysis Merck Darmstadt Germany Cat No 1 00983 1000 Ethanol 70 See section 3 below for instruction 10x EDTA running buffer for the sequencer 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 Germa
7. Ethidium bromide is potentially carcinogenic Wear appropriate protection e g nitril gloves Manual No 38 Page 8 of 16 CTS SEQUENCE HLA DQB1 Revision March 8 2012 Lot No SDQB04 0 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 with 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 340 to 660 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
8. Exon 2 DQB01 DQB1B 02 Exon 2 Black marker line Figure 1 Mix position on CTS SEQUENCE HLA DQB stripe o Sequencing primers The tubes containing the sequencing primers 500 ul have colorless caps Sequencing of exon 3 is only possible with the locus specific mix DQBO08 Table 1 Labeling of the sequencing primers HLA Locus Tube label Sequenced Applicable for Direction of Exon Mix sequencing DQB E2F 2 DQB01 DQB07 forward DQB E2R 2 DQB01 DQB07 reverse Bee DQB E3F 3 DQBO08 forward DQB E3R 3 DQBO08 reverse Manual No 38 Page 4 of 16 CTS SEQUENCE HLA DQBI Revision March 8 2012 Lot No SDQB04 0 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 38 Page 5 of 16 CTS SEQUENCE HLA DQB1 Revision March 8 2012 Lot No SDQB04 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
9. 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 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 injectio
10. agarose gel If you use CTS electrophoresis chamber and CTS combs see www ctstransplant org for order information proceed as follows 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 or a microwave oven Cool down to 60 C add 17 pl 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
11. asensssosontenndsdcwsesbescseaedevescoaansanacecsdsesedoosecsscbes connsssadscedsscwassonassoontsonnseseasoss l 5 1 Amplification ennn ee oesie aiee decade ANAE EAA E E E SEE EEEE ON aE ECESE EEE E ET amp 5 2 COL CONEPOL rrenen cents cba g ashe esas oak tng Sede saab ys anda ee be lee tee begs EAE T E EEA EE E AE Ei amp 5 3 Purification of the amplification products sssessesesseeeeeeesseeessrseresesrreresreersserrreseseresesrenreseesrnererreseset 9 5 4 SEQUENCING redetOh irige nasa EE be segues echoes E EEEE E E EEE E E E EET e ESEESE EEEE iaeoa 5 5 Purification of the sequencing productS sseessseseeseessreeseeresrssrreresrseresesresrsserrresestensesteseeseesrnsesereees 5 6 Sample preparation for sequencing runs 6 Start of a sequencing run on the sequencer sessessessesessoesesoossesoesossessossesoossesoesoeseesossesoossesoesoesessossesssssse LI 6 1 Instrument protocol for ABI Prism 3100 Genetic Analyzer sccescesceeseecceeecesecesecesecnsecseceeesseeseensees Il 6 2 R n SEQUENCIIG sr ehnan ara Seapets etek eean EEEE eaaa be ok dues a EES tae Se NEE st bass e aaas Il Te Result evalu atom ssscsciessctsssissseccdascosdesesasescdescccsasbasseavtecesudussaasessesoacvdsctaaeessoesestseassasessdssetesssscbeascencdssesesssesvees U2 8 T leshooti 13 TOU DIES OOCING a25 352255 25s coaed sadasecuctescaseesdecacccestesseacduceseceesaactseceedetevatecdeastecsascuesecsecscossadeeatesseaccspesesuasacsecseas 6 1 AMP LUfiCAT OM
12. ategorically ruled out 2 Materials and Equipment 2 1 Materials included in the CTS SEQUENCE HLA DQBI1Kit The SEQUENCE HLA DQBI Kit provides reagents sufficient for twenty four HLA DQB1 high resolution typings and contains 1 Twenty four 8 well PCR stripes with prepipetted and dried primer mixes each stripe for one HLA DQBI typing Store at 4 C in pre PCR area 2 2 tubes of CTS SEQUENCE PCR Buffer 1400 ul Store at 20 C in pre PCR area 3 Sequencing primers 500 ul each DQB E2F DQB E2R DQB E3F DQB E3R Store at 20 C in post PCR area Manual No 38 Page 3 of 16 CTS SEQUENCE HLA DQB1 Revision March 8 2012 Lot No SDQB04 0 a PCR stripes and amplification mixes The amplification primers are prepipetted and dried in colorless PCR stripes composed of 8 cavities For quality reasons we recommend to use only the caps included in the package Figure shows the positions of the PCR mixes on the stripe and the allele group s and the exons amplified by these mixes Mix DQBO to DQBO06 are for group specific amplification of exon 2 mix DQBO7 is for locus specific amplification of exon 2 and mix DQBO08 is for locus specific amplification of exon 3 Mix Amplified Allels te DQB08 All HLA DQB1 Alleles Exon 3 DQB07 All HLA DQB1 Alleles Exon 2 DQBO6 DQB1 06 01 Exon 2 DQB05 DQB1 06 06 01 not amplified Exon 2 DQB04 DQB1 05 Exon 2 DQB03 gine ae not amplified ExONE DQB02 DQB1B 03 01 01G 03 04
13. enter the reaction volume of 10 ul Proceed with the purification of the sequencing products immediately when the sequencing reaction has finished 5 5 Purification of the sequencing products Residual ddNTPs must be removed 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 pl 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 op
14. n 3 Mix DQB08 should be performed in forward and reverse direction Setting up a sequencing reaction gt Create a pipetting scheme determining which amplicon s and which sequencing primer s are pipetted into which position s of the optical 96 well reaction plate An example of a pipetting scheme can be seen in the appendix Manual No 38 Page 9 of 16 CTS SEQUENCE HLA DQB1 Revision March 8 2012 Lot No SDQB04 0 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 A 60 C 2 min a 3 4 C 2 Cave Do not forget to
15. n 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 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 38 Revision March 8 2012 Page 13 of 16 CTS SEQUENCE HLA DQB1 Lot No SDQB04 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
16. ny 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 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 Manual No 38 Revision March 8 2012 Page 6 of 16 CTS SEQUENCE HLA DQB1 Lot No SDQB04 0 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
17. oosina naa i css iesiseavdes veess ceva a SEa EaR a SS EaI EEES SER 13 8 2 NAAA AE E E 13 Appendix Worksheet Amplification Protocol cscccecscsccscccscsccscccscccsccssecssscsscssssssnees I5 Worksheet Pipetting Scheme ceccscscscscsccscccsccccccccscscscccsscsscssccssscssssssssens 16 Manual No 38 Page 2 of 16 CTS SEQUENCE HLA DQB1 Revision March 8 2012 Lot No SDQB04 0 The CTS SEQUENCE HLA DQBI 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 DQB1 with the CTS SEQUENCE HLA DQB1 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 and locus specific amplification of exon 2 of HLA DQB 1 and additionally locus specific amplification of exon 3 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 Sequencing of exon 3 helps to reduce ambiguities The SEQUENCE HLA DQBI Kit is validated and optimized with following reagents instruments softwares and methods GeneAmp PCR System 27
18. op 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 then 10 bases out of 20 have QVs less then 15 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 K Volts 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 bep Settings Sample Manager Basecaller KB bcp Dye set primer file KB_ 3100 POP6 BDTvl mob Settings Plate Record Dye Set E 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 Manual No 38 Revision March 8 2012 Page 11 of 16 CTS SEQUENCE HLA DQB1 Lot No SDQB04 0 JSI Medical Systems GmbH Kippenheim Germany see section 7 the sample naming conventions are Sample name_Amplification mix_ Sequencing primer Example Sample DQB01_ DQB E2F if amplification mix DQBO1 was used in the sequencing reaction with the DQB E2F sequencing primer 2 Place sample
19. ot No SDQB04 0
20. s 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 into 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 HLA DQBI1 Name DQB E2F DQB E2R DQB E3F DQB E3R Gene DQBI1 DQBI DQB1 DQB1 Direction fwd rev fwd rev SeqPrimer gene parts E2 E2 E3 E3 RFName DQB E2F DQB E2R DQB E3F DQB E3R Sorting 0 0 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 38 Revision March 8 2012 Page 12 of 16 CTS SEQUENCE HLA DQB1 Lot No SDQB04 0 8
21. 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 Purification 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 38 Revision March 8 2012 Page 14 of 16 CTS SEQUENCE HLA DQB1 Lot No SDQB04 0 CTS SEQUENCE HLA DQBI1 Amplification Protocol For Lot SDQB04 0 DNA No Thermocycler Date Lot Volume PCR Buffer 10 85 pl TAQ 0 15 pl DNA 25ng ul 4 ul 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 seg Pelee Amplified Allels alles DQB01
22. tical 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 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 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 Manual No 38 Page 10 of 16 CTS SEQUENCE HLA DQB1 Revision March 8 2012 Lot No SDQB04 0 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 St
23. 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 stripe into the thermocycler and start the ampli 0 15 ul Tag 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 amplification buffers often contain PCR inhibitors e g EDTA 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 i 95 C 15s 3 61 C 50s 22 72 C 1 min 30 s 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
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SDQB04 0 sdb040206 sb0402 sb0401 sb0400 sb0403-2 sb0404-10 sb0401h sb0401l sb0403tl sb040 payer id sb0404-11 sb0409-10 sb040001en sq 0406 sb0407