IFU026_PD5.2-5 SBT Resolver IFU (RUO)
Contents
1. Page 12 of 16 For Research Use Only Troubleshooting Problem Possible cause s Solution No or weak PCR product Poor quality DNA Assess DNA quality by gel electrophoresis Intact DNA should be approx 3kb with little or no evidence of smearing on gel Re extract DNA and repeat PCR where possible Insufficient quantity of DNA added to PCR Check concentration of DNA is between 20 100ng uL Re extract DNA and repeat PCR where possible Presence of PCR inhibitors in genomic DNA Avoid the use of whole blood specimens containing heparin Re extract DNA and repeat PCR where possible DNA polymerase not added to Repeat PCR Ensure the mastermix or insufficient mastermix components are mixing of mastermix prior to addition to samples added and mixed sufficiently by vortexing Thermal cycling problems Check the thermal cycling run parameters Check the run history to ensure that the run was not terminated prematurely Ensure that the thermal cycler is Operating according to manufacturer s specifications and is regularly maintained No ethidium bromide added to the gel Submerge the gel in a staining bath containing 1X TBE with 0 5mg mL ethidium bromide Destain in 1X TBE before taking gel image Ensure ethidium bromide is added to gel prior to pouring Incorrect band sizes Incorrect kit used Check that the appropriate kit is used Incorrect thermal2. 5 7 Place the inverted reaction wells and paper towel or tissue into the centrifuge Centrifuge at 350g for 1 minute to remove any residual supernatant 5 8 Remove the reaction wells from the centrifuge and place in an upright position on the work bench Discard the paper towel or tissues 5 9 Prepare fresh solution of 8096 ethanol with absolute ethanol and sterile water 5 10 Add 60uL of 80 ethanol to each well Reseal the wells and vortex briefly 5 11 Spin at 2000g for 5 minutes 5 12 Repeat steps 5 6 and 5 7 5 13 Remove the reaction wells from the centrifuge and discard the paper towel Reseal the reaction wells and proceed to the denaturation step Otherwise store at 20 C in the dark It is recommended that the extension products are run on the DNA sequencer within 24 hours of setting up the sequencing reactions Page 10 of 16 For Research Use Only 6 Denaturation amp Electrophoresis of Sequencing Reaction Products NOTE The procedure for the denaturation of extension products in Hi Di Formamide described here may not be necessary if purification procedures other than the ethanol precipitation have been used It is strongly recommended that users validate alternative procedures before proceeding 6 1 Add 12uL of Hi Di Formamide to each reaction well Vortex and centrifuge the wells plate briefly 6 2 Incubate the reaction wells at 98 C for 5 minutes Following incubation ensure that the reaction wells are cool
3. Applied Biosystems by Life Technologies before separation and detection on an automated fluorescent DNA sequencer It is recommended that the resulting data is then analysed with Assign sequence analysis software from Conexio Genomics Pty Ltd Page 3 of 16 For Research Use Only Kit Composition Kit S7 PRE PCR Contents POST PCR Contents No of vials No of vials HLA DRBl HH PDS2 5Q0 20tests DNAPOL DRBI 1x 9uL PRAA each HH PD5 2 5 50 50 tests DNA POL DRB1 1 x 18uL 1 x 110uL each The PRE PCR kit contains a vial of a locus specific PCR mix i e consisting of PCR buffer dNTPs MgCl and locus specific PCR primers along with a single vial of DNA polymerase DNA POL DRB1 The POST PCR kit contains sequencing primers e g Page 4 of 16 For Research Use Only Storage Requirements The PRE and POST PCR boxes may be separated and stored in designated PRE and POST PCR freezers When stored at 20 C temperature range of 15 C to 25 C is acceptable the kit components can be used until the expiry date indicated on the outer labels and can tolerate up to 25 freeze thaw cycles To maintain optimal kit performance the kit components should be removed from the 20 C storage location and thawed rapidly at room temperature before use The kit components with the exception of the polymerase should then be gently vortexed to ensure that the components of each
4. For Research Use Only 1 6 Seal the reaction wells Mix gently by vortexing and centrifuge briefly 1 7 Place the reaction wells into a thermal cycler and run according to the thermal cycling conditions below 95 C 10 mins 96 C 20 secs 60 C 30 secs 33 cycles 72 C 3 mins 15 C hold 1 8 Amplification takes approximately 2 5 hours to complete 1 9 When the PCR is completed remove the reaction wells plate from the thermal cycler and either proceed directly to gel electrophoresis or store at 4 C until required NOTE Purification of amplicons by ExoSap IT treatment should occur within 24 hours of completion of PCR 2 Agarose Gel Electrophoresis 2 1 Confirm successful amplification by agarose gel electrophoresis using 2uL of each PCR product combined with 5uL loading buffer alternative volumes of loading buffer should be validated prior to use The use of 1 agarose gels is recommended The number and expected sizes of the resultant amplicons will vary according to the sample genotype Expected PCR amplicon sizes range between 450 bp 850bp 3 Purification of PCR Product NOTE Purification systems other than EXOSAP IT e g Agencourt AMPure XP or column based systems can be used to purify these PCR products It is strongly recommended that users validate these procedures before proceeding If EXOSAP IT is to be used it is recommended that users follow the procedure described below 3 1 Prep
5. Resolver kits were developed and validated using the Assign SBT and Assign ATF software developed by Conexio Genomics Pty Ltd For more details please refer to the Conexio Genomics website http www conexio genomics com Page 11 of 16 For Research Use Only Performance Characteristics Conexio Genomics Pty Ltd s SBT Resolver kits are locus specific assays Sequence analysis of PCR and sequencing primer sites and performance evaluation has not identified alleles that are not amplified through the recommended use of these kits In most instances the use of the sequencing primers incorporated in each kit will produce a HLA typing for most samples without the need for further resolution In those instances where heterozygous ambiguities remain the use of resolving sequencing primers such as SBT Resolver HARPS is recommended It should be noted that mutations at amplification or sequencing primer sites are possible and may result in allele drop out Samples that suggest a homozygous typing result must be confirmed by alternative procedures Limitations and Cautions e It is strongly recommended that these kits are validated by the user prior to implementation in the laboratory using samples whose HLA type has been determined by other molecular based procedures In particular any deviations from this procedure e g the use of alternative PCR or DNA sequencing purification procedures must be validated by the user prior to
6. Temperature and time 25 96 C 10sec 50 C 5sec 60 C 2min 1 4 C hold 4 8 Once the program is complete remove the reaction wells plate from the thermal cycler and either proceed directly to purification of the reaction products or store in the dark at 4 C until required It is recommended that samples are purified and run on the DNA sequencer within 24 hours 5 Purification of Sequencing Reaction Products NOTE Purification of the reaction products may be carried out by procedures other than the ethanol precipitation method described here It is strongly recommended that users validate these procedures before proceeding 5 1 Briefly centrifuge the reaction wells plates before proceeding If reusable lids caps have been used during thermal cycling label the lids caps to avoid cross contamination 5 2 Carefully remove the seals 5 3 To each reaction well add 5uL of 125mM EDTA pH8 0 Ensure that the EDTA reaches the base of the reaction well 5 4 Add 60 uL of 100 ethanol to each reaction well Seal the wells plate and vortex briefly but thoroughly to ensure thorough mixing 5 5 Pellet the extension products by centrifuging at 2000g for 45 minutes IMMEDIATELY PROCEED TO THE NEXT STEP If this is not possible re centrifuge for an additional 10 minutes before proceeding 5 6 Remove the seals to the reaction wells and discard the supernatant by inverting the reaction wells onto paper towel or tissues
7. bee ERE Gu cau educ e SEE EA 9 5 PURIFICATION OF SEQUENCING REACTION PRODUCTS cesses eene eren nnns enn 10 6 DENATURATION amp ELECTROPHORESIS OF SEQUENCING REACTION PRODUCTS 11 T EDITING AND ANALYSIS OF ELECTROPHEROGRAMSG ssssssssescceeseseeaesecececsesesaeseeecsceeseaasaeeeesceeees 11 PERFORMANCE CHARACTERISTICS cresce ee eere eee tn nee ea tese e enne eese tn Peste tee seen Pese ee tn nee ta 12 LIMITATIONS AND CAUTIQONS ceo scares to eo ih eoe nora have h oe nere sob on koe ode n eR essri sss ssas toss reis aee dn 12 LICENSE OR YQ 12 cegdmogste uuo E 13 RELATED QD OE 15 SUPPORT AND CONTACT DETAJTLS ssec er seco oo een oP oer pee EF ee on eee Irae ao Ee as sissi SEES EVER eS eR Sea ro Page 16 Page 2 of 16 For Research Use Only Principle The HLA Sequence Based Typing SBT procedure described here involves the initial amplification of the target sequences followed by treatment with ExoSAP IT to remove unincorporated primers and dNTPs The amplicon is then used as a template for direct automated fluorescent DNA sequencing using customized sequencing primers and the Big Dye Terminator sequencing chemistry available from Applied Biosystems by Life Technologies The extension products are purified according to the ethanol precipitation method and denatured using Hi Di formamide available from
8. implementation e These kits have been validated using panels of samples whose genotypes cover a broad range of alleles However it should be noted that rare alleles and alleles with polymorphisms in amplification and sequencing primer sites may be encountered and these may not be amplified or sequenced e The nature of HLA sequence based typing is such that factors other than the PCR mix may result in preferential amplification or allele drop out As a consequence apparent homozygous typing results should be confirmed using alternative methods and or family genotyping e A positive control human DNA and negative control sterile water must be included on every PCR run The positive control must produce a PCR product of the appropriate size depending on the locus amplified and the resultant sequence must be in concordance with the sample s genotype There must be no PCR products in the negative template control for each experiment If a band is evident contamination may have occurred at some level and the run must be repeated e Occasionally there may be additional fainter PCR products evident These additional bands do not interfere with sequence results or quality License The SBT Resolver kits contain GoTaq Hot Start Polymerase DNA POL which is manufactured by Promega Corporation for distribution by Conexio Genomics Pty Ltd Licensed to Promega under U S Patent Nos 5 338 671 and 5 587 287 and their corresponding foreign patents
9. provided for use with the HH PD5 2 5 kits RB TG344 R is a HARP directed to the codon 86 dimorphism Its use is optional 4 2 Prepare a fresh solution of sequencing primer mix on ice each time a sequence reaction is performed The composition and volumes for the mix indicated below are per sample Component Volume Sequencing primer 2uL Sterile water 11 5 uL BigDye Terminators luL 5x Seq Rxn Buffer 3 5 uL 4 3 Mix each sequencing reaction mixture gently by pulse vortexing 4 4 Dispense 18uL of the sequencing reaction mix into each appropriate reaction well NOTE For runs which involve few samples with many sequencing primers it is acceptable to dispense the sequencing primer 2uL directly into the individual reaction wells A master mix may then be created composing of sterile water BigDye Terminators and 5x Seq Rxn Buffer of which 16uL is to be dispensed into each reaction well It is strongly recommended that use of this alternative procedure is validated by the user prior to implementation 4 5 Add 2uL of purified PCR product to each appropriate well NOTE Care must be taken to prevent cross contamination of sequence reactions 4 6 Seal the reaction wells mix gently and centrifuge briefly to ensure that the contents are located at the base of each reaction well 4 7 Place the reaction wells into a thermal cycler and run according to the following profile Page 9 of 16 For Research Use Only Number of cycles
10. 50 HH PD10 1 20 HH PD10 1 50 AN PD11 0 0 20 AN PD11 0 0 50 AN PD12 0 0 20 AN PD12 0 0 50 AN PD13 0 0 20 AN PD13 0 0 50 LC PD2 9 20 LC PD2 9 50 Product code CGX0036 SBT Resolver HLA B kit 20 and 50 tests SBT Resolver HLA C kit 20 and 50 tests SBT Resolver HLA DQBI kit 20 and 50 tests SBT Resolver HLA DPB1 kit 20 and 50 tests SBT Resolver HLA DRB3 kit 20 and 50 tests SBT Resolver HLA DRB4 kit 20 and 50 tests SBT Resolver HLA DRB5 kit 20 and 50 tests SBT Resolver HLA B57 kit 20 and 50 tests SBT RESOLVER aes Product codes C1 TT98 F 20 C1 CT102 F 20 C1 GG307 R 20 C1 TA368 F 20 C1 BTA F 20 C1 GA206 F 20 C1 AG360 F 20 C1 CC486 F 20 C1 CG572 R 20 C1 CT97 F 20 C1 GC302 R 20 C1 GG539 R 20 RB 01 F 20 C1 AC98 F 20 C1 CC102 F 20 C1 GG363 AF 20 C1 GT355 R 20 C1 BCG F 20 C1 CG319 F 20 C1 GC363 F 20 C1 CT559 R 20 C1 GAG601 R 20 C1 CT112 F 20 C1 CG343 F 20 C1 AA601 R 20 RB 04 F 20 C1 TC98 F 20 C1 AG203 F 20 CI TA363 F 20 C1 GG362 R 20 C1 CC144 F 20 C1 CA309 R 20 C1 GG363 BF 20 C1 GA559 R 20 C1 CG134 F 20 C1 CA343 F 20 RB 09 F 20 Page 15 of 16 CI TA98 F 20 C1 GT240 F 20 C1 AT362 F 20 C1 CT423 F 20 C1 AC206 F 20 C1 GAT309 R 20 C1 TA420 F 20 C1 AC559 R 20 C1 AG270 F 20 C1 GA361 F 20 RB 15 F 20 C1 CA102 F 20 CI TT368 F 20 C1 AC497 F 20 C1 CG570 R 20 C1 GC209 F 20
11. C1 GAA309 R 20 C1 AC362 F 20 C1 GG572 R 20 C1 AC302 R 20 C1 TG539 R 20 RB 52 F 20 For Research Use Only RB GG125 F 20 RB TA164 F 20 RB AT257 R 20 QB TA173 F 20 PB AT251 R 20 RB AA197 F 20 RB TT227 F 20 RB TT321 R 20 QB CT173 F 20 PB GT313 R 20 RB TT197 F 20 RB AT258 F 20 RB GT344 R 20 QB TA185 F 20 PB TACI21 F 20 RB GT196 F 20 RB GC258 F 20 RB TG344 R 20 QB CG353 R 20 PB GG341 R 20 RB GA196 F 20 RB CT257 R 20 QB GG353 R 20 PB GC194 F 20 PB AG341 R 20 General Purpose Laboratory Reagents MgCl2 1 0 50 2mM MgCl MgCl2 1 0 3000 SEQ BUF 2 0 400 5x Seq Rxn Buffer SEQ BUF 2 0 5000 EDTA 3 0 200 EDTA 3 0 5000 125mM EDTA pH8 0 Please contact your local distributor for further details Support and Contact Details Conexio Genomics Pty Ltd 8 31 Pakenham St Fremantle 6160 Western Australia Tel 61 422 863 227 email support conexio genomics com Skype conexiocgx Website www conexio genomics com Or your local distributor For ordering details please refer to the Olerup website http www olerup com Conexio and HARPS are trademarks of Conexio 4 Pty Ltd HARPS is a registered trademark in some territories Page 16 of 16 For Research Use Only
12. CONEXIO SBT RESOLVE Rose PCR Amplification and Sequencing of HLA DRB1 Full Exon 2 Instructions for Use Version No 1 0 Issue Date August 2012 For Research Use Only Not for use in diagnostic procedures No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease Conexio Genomics Pty Ltd 8 31 Pakenham St Fremantle 6160 Western Australia Australia Page 1 of 16 For Research Use Only Contents d Edd ug DOS 3 KIFT COMPOSITION gr 4 STORAGE REQUIREMENTS sss cssssssscssevssssnsessesocssavssvssosnscevecssbnsseesasectcsecsesnastbonsesantenieassoncsevcasensesesces 5 MATERIALS REAGENTS AND EQUIPMENT NOT SUPPLIED seoessseessccecsseceessecescseessooeessese 5 SAMPLE REQUIREMENTS siisesiscccdsess sossssssessseeddensesoshcodaussdeassovescoedeeddeesoosascocessogessentsantsesoas vnssessscesesess 6 WARNINGS AND SAFETY PRECAUTIONS ccssscsssssssscsssccssssssscesssssssccsssessssscssessssssssessssces 6 PRO CEDUREivivscscscesisscsssnssse uessectscstessoatsntesssnateesstveneessexdsesaceveedesaatcuesesSuatenedsdsunsseaxesdaccsvetesessvsbuessexctecsess 7 1 PCR oett ete fere ea Goes cen eee d secreti test cate cede ER vende 7 2 AGAROSE GEL ELECTROPHORESIS siiis liring a o lanit iii aN e iae aat iaia 8 3 PURIFICATION OE PCR PRODUCT rer i so Fa aene aea ena i i iih 8 4 SEQUENCING REAGCTION i secede doce e nee dee dues a ege iden e
13. are a mastermix consisting of 4uL of ExoSAP IT and 8uL of 2mM MgCl per sample to be purified Dispense 12uL of the mastermix into the reaction well of each reactive sample Seal the wells and vortex to mix Centrifuge briefly before placing into the thermal cycler Run the thermal cycler according to the following profile 37 C 30mins 80 C 15mins 4 C hold 3 2 Upon completion dilute the purified product 1 4 with sterile water This dilution step will ensure that there is sufficient template to perform the sequencing reactions and ensure that the concentration of the template is sufficient to produce good quality sequence data NOTE A higher dilution factor e g 1 8 may be required if consistently high signals and associated noise and artefacts are observed Weaker PCR products may require a lower dilution factor Page 8 of 16 For Research Use Only 3 3 ExoSAP IT treated samples may be stored at 4 C until ready for use ExoSAP IT treated samples can be stored at 4 C for up to a week before use but should be stored at 20 C for long term storage 4 Sequencing Reaction NOTE In instances where heterozygous ambiguities are to be resolved with hemizygous sequencing primers such as HARPS please refer to the SBT Resolver HARPS Instructions for Use 4 1 Table 2 lists the sequencing primers that are to be used for this kit HLA DRBI DRB1EX2F DRB1EX2R 2 DRB1EX3R 2 RB TG344 R Table 2 Sequencing primers
14. ble e Pre and Post PCR activities must be strictly physically separated Use specifically designated equipment reagents and laboratory coats e Ethidium bromide is a potential carcinogen Protective gloves must always be used when preparing and handling gels Dispose of ethidium bromide gels and buffers according to local and national guidelines e While viewing and photographing agarose gels under UV light always avoid direct exposure and use appropriate UV blocking face protection disposable gloves and laboratory coats Procedure 1 PCR 1 1 A separate PCR reaction will need to be set up for each sample to be tested Each run should include appropriate positive control s of known genotype and at least one negative control 1 2 Prepare a fresh solution of PCR master mix each time a PCR is performed Quickly thaw the locus specific PCR mix at room temperature Once thawed vortex briefly 1 3 Dispense the required volume of PCR mix and DNA polymerase into a sterile tube for the number of samples to be tested refer to Table 1 below for the volume per reaction Pulse vortex the solution 3 4 times Component Volume DNA POL DRB1 0 3uL Table 1 Composition of the master mix required per sample 1 4 Dispense 17uL of the master mix into each reaction well 1 5 Add 3uL of sample DNA or appropriate positive control s to each reaction well Add 3uL of sterile water to the negative control reaction well Page 7 of 16
15. ed quickly to room temperature e g place on ice or use the thermal cycler to perform the denaturation and cooling steps before being placed on the sequencer If it is not possible to run the plates immediately store at 4 C until required NOTE Ensure that there are no air bubbles in the reaction wells These can enter and damage the capillary 6 3 Load the reaction wells plate onto the automated sequencer and prepare the data collection file according to the sequencer manufacturer specifications 6 4 The following instrument parameters have been validated by the manufacturer using Big Dye Terminator Sequencing Kit v3 1 and POP 7 These parameters may require user validation for other polymers sequencing chemistries and instruments Please refer to the appropriate instrument user s manual for detailed instructions and guidance e g ensure that the dye set setting is appropriate for the chemistry used for example v1 1 Big Dye Terminator sequencing chemistry will require a different dye set Parameter Setting Dye set Z_BigDyeV3 Mobility file KB_3730_POP7_BDTV3 Basecaller KB bcp Run Module Regular FastSeq50_POP7 Injection time 15 sec Run time 3000 sec 6 5 Use the instrument s data collection software to process the raw collected data and create the sequence files Please refer to the appropriate instrument user s manual for detailed instructions and guidance 7 Editing and analysis of electropherograms The SBT
16. nd listed above Amplification of closely related HLA genes Check thermal cycling parameters Consider adjusting the parameters if an alternative thermal cycler is used Poor PCR purification Ensure ExoSAP IT treatment is undertaken according to kit s user instructions Ensure that the PCR mixture is mixed thoroughly with ExoSAP IT Contaminated reactions sequencing Ensure that all steps are taken to prevent cross contamination Change pipette tips wherever possible Add liquids at the top of the reaction wells Prevent aerosols Contaminated sequencing primer Check sequence quality of the other sequencing primers and other samples using the same primer Contaminated dye terminator mix or sequencing buffer Repeat sequencing with fresh aliquot of reagents Poor purification of sequencing products Repeat sequencing and ensure that purification is undertaken according to manufacturer s instructions Page 14 of 16 For Research Use Only Presence of Dye blobs products Poor purification of sequencing kit instructions Purify products according to Ensure products are washed sufficiently with 80 ethanol Related Products ASSIGN stas SBT RESOLVER SBT Resolver HLA A kit 20 and 50 tests XH PD1 1 2 20 XH PD1 1 2 50 BS PD2 1 2 20 BS PD2 1 2 50 HH PD3 2 2 20 HH PD3 2 2 50 PQ PD6 2 2 20 PQ PD6 2 2
17. program used cycling Check the parameters thermal cycle PCR contamination Check the negative control for evidence of contamination Decontaminate work area and repeat PCR Repeat PCR to identify source of contamination Consider using a fresh kit If the genomic DNA of a sample appears to be contaminated re extract or Page 13 of 16 For Research Use Only obtain an alternative source of DNA Weak signal intensity of electropherograms Weak PCR product Check gel image Sequencing weak PCR bands is NOT recommended as the sequence quality may be insufficient for SBT Consider using a lower dilution factor eg 1 2 1 3 after PCR purification Insufficient reaction products applied to sequencer Check sequencer parameters Injection time and voltage may need to be increased Problems during purification of sequencer products Use extreme care when discarding the supernatant as it may dislodge the pellet Signal intensity is too Too much PCR product Check the gel image Consider high Presence of high using a higher dilution factor fluorescent peaks following PCR purification artefacts Check the amount of DNA polymerase used in the PCR Too much reaction products Check instrument parameters applied to sequencer Consider reducing the injection time and voltage Noisy baseline high Contaminated PCR product Refer to corrective actions backgrou
18. rior to use Page 5 of 16 For Research Use Only Sequencing Reaction 17 18 BigDye Terminator Cycle Sequencing Kit v3 1 or v1 1 Applied Biosystems by Life Technologies 5x Sequencing Reaction Buffer Conexio Genomics product code SEQ BUF 2 0 400 or SEQ BUF 2 0 5000 or BigDye Terminator v3 1 or vl 5X Sequencing Buffer Applied Biosystems by Life Technologies Purification of Sequencing Reaction Products 19 20 125mM EDTA pH8 0 available for purchase from Conexio Genomics product code EDTA 3 0 200 or EDTA 3 0 5000 Absolute and 80 Ethanol Each run requires freshly prepared 80 ethanol consisting of absolute ethanol and sterile water DO NOT USE DENATURED ETHANOL The use of alternative sequencing purification techniques requires validation by the user prior to use Denaturation and Electrophoresis of Sequencing Reaction Products 21 22 23 Hi Di Formamide Applied Biosystems by Life Technologies product code 4311320 Automated DNA Sequencer and accessories e g Applied Biosystems by Life Technologies ABI Prism 3730 including data collection and software These kits have been tested and validated on the Applied Biosystems by Life Technologies 3100 3730 and 3730xl capillary sequencers and software The use of other denaturation techniques and sequencing platforms requires validation by the user prior to use HLA Sequencing Analysis Software e g A
19. ssign SBT version 3 5 or higher or Assign ATF Conexio Genomics Pty Ltd Sample Requirements 1 Sterile water negative no template control 2 High molecular weight human genomic DNA concentration range of 20 100ng uL in Tris EDTA buffer and OD sooso 1 8 extracted from ACD or EDTA anticoagulated whole blood specimens Do NOT use whole blood specimens containing heparin ome and Safety Precautions This kit must be used by trained and authorized laboratory personnel All samples equipment and reagents must be handled in accordance with good laboratory practice In particular all patient material should be considered as potentially infectious The use of gloves and laboratory coats is strongly Page 6 of 16 For Research Use Only recommended Handle and dispose of all sample material according to local and national regulatory guidelines e There are NO dangerous substances contained in any of the kit components e Do NOT use reagents beyond their expiration date e The use of kit components from different kit batches is NOT recommended Such use may affect the assay s performance e Use of reagents not included in this kit or not listed under Materials Reagents and Equipment Not Supplied e g alternative Tag DNA polymerases is NOT recommended Such use may affect the performance of the assay e Care should be taken to prevent cross contamination of DNA specimens Change tips between DNA specimens wherever possi
20. tube are appropriately mixed after thawing After use the kits components should be returned immediately to 20 C Materials Reagents and Equipment Not Supplied PCR 1 Sterile water 2 Electronic or mechanical pipettes and aerosol resistant tips 3 Thermal cycler with heated lid These kits have been validated using the following thermal cyclers MJ Research PTC 225 DNA Engine DYAD Applied Biosystems by Life Technologies Gene Amp PCR System 9700 and Eppendorf Mastercycler Pro Use of other thermal cyclers with these kits requires validation by the user 4 0 2mL thin walled thermal cycling reaction tubes 8 well strips or 96 well plates Use those recommended for use with your thermal cycler 5 Sterile 1 5mL tubes 6 Sterile work area such as biological safety cabinet or hood T Table top centrifuge with plate adapters and capacity to reach 2500 x g 8 Vortex Agarose Gel Electrophoresis 9 Agarose gel electrophoresis apparatus 10 1 agarose molecular biology grade TBE gel containing 0 lug mL ethidium bromide 11 Loading buffer 12 PCR Marker suitable to cover range of 300 1300 bp 13 UV transilluminator Purification of PCR Product 14 ExoSAP IT USB Products Cat No 78200 for 100 reactions 15 2mM MgCl available for purchase from Conexio Genomics product code MgCI2 1 0 50 or MgCI2 1 0 3000 16 Shaker The use of alternative PCR purification techniques requires validation by the user p
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