SB05-0
Contents
1. 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 I bases on amplification and sequencing primers which are located outside exon 2 and 3 For many HLA class I variants only the sequence of the antigen recognition site exon 2 and 3 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 B Kit The SEQUENCE HLA B Kit provides reagents sufficient for twenty four HLA B high resolution typings an2. Working Instruction CTS SEQUENCE HLA B For high resolution typing of HLA B Product No 334 Lot No SB05 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 34 Page of 16 CTS SEQUENCE HLA B Revision August 04 2014 Lot No SB05 0 Content 1 TNMthOGUCHON sacsscce sccs ceccecensvesesvesbccnssssvecassessecedsesuatssvesulsedeeveodavbestosecdetesdeseodecensuesbosuetessesntessvessstesslanssesseccssesses D 2 Materials and Equipment cscscsscsssesscessessecesesesees 2 1 Materials included in the CTS SEQUENCE HLA B Kit ccccccccssscessseeeseeensecenseceeaceceaceeeaeecsseeenaeceeeeeeaes 3 2 2 Storage GA EXPIT ATION iis cdi Acie ests a Ta EE AEN NEET REEE AEV EAA EEE N RAT 3 2 3 Materials and equipment not inCluded ccsccscssccssesesscnsessecseesecseeecseeseeseseescsseeesenseesesaecaeesesneeaeeseeneeeas 5 3 Preparation of buffers and agarose gel ssscscscscscscececsssssccsssessscsecssessecssecsesccscssscsssssssscsssesesssssseseeseees 4 Isolation and concentration measurement Of DNA ssscccssssssccsssccsscssccssssccccssssccscsssccsssssscccssssccccsscces 7 5 Test procedure siccssssstsiccssisissessenksscvsessasssseseccndssecvessinsSesceaswansosoasonesscensedoasectsceestesesoasanasscsdeesessssensecaseseeas
3. 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 Dye set primer file KB_3100_POP6_BDTv1 mob Settings Plate Record Dye Set E Run Module CTS2600 Mobility File 3100_POP6_BDTv1 mob Manual No 34 Revision August 04 2014 Page 11 of 16 CTS SEQUENCE HLA B Lot No SB05 0 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_B01_B E2F if amplification mix BO1 was used in the sequencing reaction with the B 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 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
4. and a mix up of samples Manual No 34 Page 8 of 16 CTS SEQUENCE HLA B Revision August 04 2014 Lot No SB05 0 vyv WV 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 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 00 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 I exon 2 and exon 3 must be completely sequenced If an allele is not separated by amplification e i if only the locus specific mix is positive or if only one of the group specific mixes the locus specific mix are positive we recommend to sequence the locus specific mix B08 in both directions forward and reverse to optimize base calling and to reduce the risk of allelic drop out If the alleles are separated by amplification e i if two group specific mixes are positive it is suf
5. 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 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 Nitril gloves Examination 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 a
6. CE HLA B Revision August 04 2014 Lot No SB05 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 B with the CTS SEQUENCE HLA B 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 exon 3 and exon 4 of HLA B genes second the amplification products are sequenced in forward and reverse direction Matching for exon 2 and 3 antigen recognition site at allele level is considered relevant in hematopoietic stem cell transplantation Sequencing of exon 4 helps to reduce ambiguities Furthermore many null alleles not expressed alleles e g B 51 11N which occur at relative high frequencies in specific haplotypes can be detected by the sequence of exon 4 The SEQUENCE HLA B 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
7. Labeling of the sequencing primers HLA Locus Tube label Sequenced Exon Direction of sequencing B E2F 2 forward B E2R 2 reverse B E3F 3 forward ae B E3R 3 reverse B E4F 4 forward B E4R 4 reverse Manual No 34 Page 4 of 16 CTS SEQUENCE HLA B Revision August 04 2014 Lot No SB05 0 Special notes In the following rather uncommon combinations of alleles allele groups ambiguities can be further resolved by using additional group specific sequencing primers not included in this kit for sequencing reactions Positive Group specific Combination amplification mix sequencing Order number used for primers sequencing Mix B06 B E2R35 433 35 51 P 5 or B E2R51 ee mix B08 B E3F35 B E3F51 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 reagent to make aliquots of appropriate volumes and store them as indicated 2 3 Materials and equipment not included Table 2 Pre PCR area s and should be avoided It is recommended Reagents materials softwares Company Catalogue number Taq DNA Polymerase 5 U ul 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 C
8. 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 B B E2F_ B E2R B E3F_ B E3R Gene Be SB Be BS B _ B Sorting Of jo o jo 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 34 Page 12 of 16 CTS SEQUENCE HLA B Revision August 04 2014 Lot No SB05 0 8 Troubleshooting 8 1 Amplification Observation Possible Cause s Solution No weak or non specific PCR product s 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 Degraded DNA New extraction of DNA DNA concentration to low New extraction of DNA DNA contains PCR inhibitors Heparinized blood New extraction of DNA Thermocycler is defect Check cycler e g with the CTS Cycler Control Kit Incorrect thermocycler program Correct programm and repeat PCR The
9. alidated 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 B 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 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 B 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 Pip
10. amber 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 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 1300 2000 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 C Interpretation hints Some mixes may occasionally show faint non specific amplifications which do not affect the sequencing results 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
11. at No E1510 10ML Magnetic stirring hotplate or a microwave oven for gel preperation Pipettes and filter tips for 0 5 10 ul 10 200 ul and 200 1000 ul volumes Eppendorf Wessing Berzdorf Germany Sequence Pilot HLA SBT JSI Medical Systems GmbH Kippenheim Germany 50x TAE buffer Photometer for spectral measurememnt of DNA concentration Inno Train Kronberg Taunus Germany Cat No GX12765 Analytical balance Manual No 34 Revision August 04 2014 Page 5 of 16 CTS SEQUENCE HLA B Lot No SB05 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
12. d contains 1 Twenty four 8 well PCR stripes with prepipetted and dried primer mixes each stripe for one HLA B 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 B E2F B E2R B E3F B E3R B E4F B E4R Store at 20 C in post PCR area Manual No 34 Page 3 of 16 CTS SEQUENCE HLA B Revision August 04 2014 Lot No SB05 0 a PCR stripes and amplification mixes The amplification primers are prepipetted and dried in blue 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 amplified by each mix Mix B01 to B07 are group specific mix B08 is locus specific B08 Mix Amplified Allels B08 All HLA B Alleles BO7 B 44 ex B 44 27 BO6 B 35 51 52 53 58 78 81 03 an B05 B 18 27 37 40 02 40 06 47 82 02 B04 B 15 46 wo jo X BO3 B 13 57 81 01 02 B02 B 08 14 38 39 42 67 73 B01 B 07 40 01 48 81 01 02 Cave Mix B06 amplifies B 35 43 weakly Black marker line Figure 1 Mix position on CTS SEQUENCE HLA B stripe z Sequencing primers The tubes containing the sequencing primers 500 ul have blue colored caps Sequencing of exon 4 is only possible with the locus specific mixes mix B08 Table 1
13. e 9 of 16 CTS SEQUENCE HLA B Revision August 04 2014 Lot No SB05 0 Table 5 Sequencing strategy recommended for HLA B example One group specific Iwo eroup speditic Only mix e g mix B01 ee Exon to ne e g mix B01 mix B02 locus specific mix and Amplification mix be se z p and Mix B08 locus specific mix r quenced igs locus specific mix positive mix B08 mix B08 positive positive Group specific mix 1 Exon 2 B E2R B E2R e g mix B01 Exon 3 B E3R B E3R Group specific mix2 Exon 2 B E2R e g mix B02 Exon 3 B E3R Exon 2 B E2F and B E2R B E2F and B E2R B E2R bopa sperie Mik eeu B E3F and B E3R B E3F and B E3R B E3R mix B08 Exon 4 B E4F and B E4R B E4F and B E4R B E4F and B E4R Thermocycler program for sequencing reaction CTS SEQ Step Temperature Time Numbers of cycles 1 96 C 1 min 1 96 C 10s 2 60 C 2 min 29 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 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 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 lo
14. ell reaction plate 1 2 4 5 6 7 8 9 10 11 12 A Stan_B01 _B E2R B Stan_B02 _B E2R c Stan_B01 _B E3R D Stan_B02 _B E3R E Stan _B08 _B E2F F Stan_B08 _B E3F G Stan _B08 _B E4F Stan_B08 H _B E4R DNA sample ID Name e g Stan Amplification pattern of the B locus amp B08 positive 308189 0 amp B02 positive Q BO1 positive Manual No 34 Revision August 04 2014 Position on plate Al A2 A3 A4 A5 A6 A7 A8 Page 16 of 16 Mix B01 was sequenced with the B E2R sequencing primer Mix B02 was sequenced with the B E2R sequencing primer Mix B01 was sequenced with the B E3R sequencing primer Mix B02 was sequenced with the B E3R sequencing primer Mix B04 was sequenced with the B E2F sequencing primer Mix B04 was sequenced with the B E3F sequencing primer Mix B04 was sequenced with the B E4F sequencing primer Mix B04 was sequenced with the B E4R sequencing primer CTS SEQUENCE HLA B Lot No SB05 0
15. eooassoes 1 5 1 Amplification monennen n o e ea ated bane EEEE O OEA E ented EA di EE A 7 3 2 COL CONITON riene nene eE sed sath aE NEEE E Mitel a AAE E E E OA AEE IE E EN 8 5 3 Purification of the amplification products seseeeeseesseseeseeseersessrsresrssrerresesresrrrresrsseerrsserreseseeereseeses amp 5 4 Sequencing TEACEION aiino ee a eE aE E EEEE Eae RENEE AE Seek wwe EE A AE E E E Neas 5 5 Purification of the sequencing products 5 6 Sample preparation for sequencing runs 6 Start of a sequencing run on the sequencer ssesessoesesoessesossesocssossesoossesoesoeseesossesoesesesesoesessossessossesoesssosss LL 6 1 Instrument protocol for ABI Prism 3100 Genetic ANdlyZer sssessesesesseseeeereeereresessrrereserreeresreseseres Il 6 2 Run SCGQUCNCING sicics ns cate de deat sus Ries eivahaig ite Sect budedocuptns Sedge op eubebuate led tdes cecuebinaedue cvsbsekbededetasued aE ees 12 7 TF AE ET i S A S E E E EE E RN A 2 S Troubleshooting ssssccsessssscssssscsssnssesssscescsssessesensseessscessessescsssnsssscsssscssessssesssesssssessessessessesscessesoes LO 8 1 AMPpUNCANON neien r a e a i e ae e Sach ea E Ea castes E eaoaai 13 8 2 DCQUENICIIG EA E EE EEEE E 13 Appendix Worksheet Amplification Protocol esssoesssssossssoossssssssssoossssosessossssssoossosoeses 14 Worksheet Pipetting Scheme seesssoesssoossssosssssoossssossssoosssssoossssosessossssssssssss 15 Manual No 34 Page 2 of 16 CTS SEQUEN
16. equencing products are quite stable when kept in the dark Manual No 34 Revision August 04 2014 Page 10 of 16 CTS SEQUENCE HLA B Lot No SB05 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 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 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 KVolts Pre Run Time 180 sec Injection Voltage 1
17. ette 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 4ul DNA 25 ng ul gt Put the minitray into the thermocycler and start the ampli 0 15 ul Tag Polymerase fication program CTS AMP see below 15 ul reaction volume Manual No 34 Page 7 of 16 CTS SEQUENCE HLA B Revision August 04 2014 Lot No SB05 0 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 Z 65 C 2 min 19 95 C 15s 3 61 C 50 s 22 122C 1 min 30s 4 4 C 0 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 ch
18. ficient to sequence the positive amplicons in only one direction we recommend to use the reverse primers Sequencing of exon 4 always use locus specific amplicons for this step should be performed in forward and reverse direction Table 5 exemplifies which sequencing primers should be used depending on positive amplification patterns 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 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 Manual No 34 Pag
19. garose gel If you use CTS electrophoresis chamber and CTS combs see www ctstransplant org for order information proceed as follows Manual No 34 Revision August 04 2014 Page 6 of 16 CTS SEQUENCE HLA B Lot No SB05 0 Add 7 g of agarose and 7 ml of 50x TAE buffer to 350 ml of ddH30 Boil to dissolve the agarose using a magnetic stirring hot plate or 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 v
20. ong 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 Page 13 of 16 Reduce the amount of PCR product used in the sequencing reaction The CTS SEQUENCE HLA B Lot No SB05 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 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 i
21. rmocycler 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 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 Manual No 34 Revision August 04 2014 Wr
22. s 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 34 Revision August 04 2014 Page 14 of 16 CTS SEQUENCE HLA B Lot No SB05 0 CTS SEQUENCE HLA B Amplification Protocol For Lot SB05 0 DNA No Thermocycler Date Lot Volume PCR Buffer 10 85 ul TAQ 0 15 ul DNA 25ng nl 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 eal Fes Amplified Allels aa Bot 1500 bp B 07 40 01 48 81 01 02 2 3 B02 1600bp B 08 14 38 39 42 67 73 2 3 B03 2100bp B 13 57 81 01 02 2 3 B04 2000bp B 15 46 2 3 B05 2000bp B 18 27 37 40 02 40 06 47 82 02 2 3 B06 1500bp B 35 51 52 53 58 78 81 03 2 3 B07 1500bp B 44 ex B 44 27 2 3 B08 1800bp All HLA B Alleles 2 4 Cave Mix B06 amplifies B 35 43 weakly B 44 27 is not amplified by Mix B07 Comment Date Signature Operator Date Signature Reviewer Manual No 34 Revision August 04 2014 Page 15 of 16 CTS SEQUENCE HLA B Lot No SB05 0 Pipetting scheme Example Optical 96 w
23. ss 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 Add 75 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 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 s
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