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Onerxn One96-wellplate(100rxns) RESGENSPECIFICPRIMERS: Program:92C(30"),56C(45"),72C(3"30")/36cycles Forwardprimer(20uM) 5 - Reverseprimer(20uM) 5 - 10XPCRbuffer(PerkinElmer) 10 1000 MgCl2(25mM) 8 800 100XdNTPs(25mMeach) 1 100 YeastgenomicDNA(0.2ug/uL) 0.2 20 ddH2O 70.45 7045 AmpliTaq(5U/uL) 0.35 35 ---- ---- 100uL 88uLaliquots . RESGENUNIVERSALPRIMERS Program:92C(30"),56C(45"),72C(3"30")/25cycles Forwardprimer(30uM) 3 300 5"ggaattccagctgaccacc3" Reverseprimer(30uM) 3 300 5"gatccccgggaattgccatg3" 10XPCRbuffer(PerkinElmer) 10 1000 MgCl2(25mM) 8 800 100XdNTPs(25mMeach) 1 100 YeastORFDNA(0.5ng/uL) 4 - ddH2O 73.6 7360 AmpliTaq(5U/uL) 0.4 40 ---- ---- 100uL 88uLaliquots GELELECTROPHORESISOFPCRREACTIONS(qualitycontrol)
Agarosegel: 1%agarose,1XTAE,0.5mg/mLethidiumbromide Buffer: 1XTAE,0.5mg/mLethidiumbromide LoADIngdye(6X): 15%Ficoll-400,0.25%xylenecyanolFF,0.25%bromophenolblue DNAsizeladder: 4mL1XTEbuffer,1mL1kbladder,1mL6Xloadingdye 1. Startwith3uLofPCRreactionsinPCRplates,afterremainderistransferredtoU-bottomplates(seenextsection). 2. Pourgelwithfourcombsof26wellseach. 3. Add1uL6XloadingdyetoPCRreactionsinPCRplates. 4. Load6uLDNAsizeladderinlane#1ofeachrow. 5. Usinga12-channelpipettor,loadsamplesA1-A12intoalternatinglanes2,4,...,24. 6. LoadsamplesB1-B12intoalternatinglanes3,5,...,25. 7. Repeatthisprocedurefortheremainingsamples,suchthattwosequentialrowsofPCRreactionsareloadedintoasinglerowofwellsinalternatinglanes. 8. Runat70-80Vuntilthefirstdyeband(XCFF)ishalfwaytothenextrowofwells. 9. Takeahigh(~1")andlow(~6/30")exposurephotographs. ComparetopredictedORFsizesandforthepresenceofsignificantdoublets. 10. RepeatPCRrxnsforfailedORFs.NOTE: For2ndPCRattempt,sortfailuresbygenesize,doublets,etc.,andmodifyreactionconditionsaccordingly. ForgenesthatstillgivePCRfailures,designnewprimers,e.g.toamplifysubregionsofgenes. DNAPRECIPITATIONS=>SeeComparisonofPCRCleanupProtocols
1. TransferPCRreactionsto96-wellU-bottomtissuecultureplates(Costar#3790). Transfer3uLbacktoPCRplatesforcheckgels(seeabove). 2. DrydownvolumeinU-bottomplatesto~50uL.(Hightemp.speecvacuumfor1hrfor8plates.) Thedryingwillbeuneven,withwellsaroundtheedgesexperiencingmoreevaporation.1hrgetsallthewellsdownto~50uL. 3. Add1/10vol.3Msodiumacetate(pH5.2)+2.5volumesethanol. Storeat-20Cforafewhourstoovernight. 4. CentrifugeinSorvallRC-3Bat3500rpmfor1hr(H-6000Arotor,RCF=3565g). 5. Removesupernatantwith12-channelaspirator(Wheaton/PGCScientifics#851388). 6. Add100uLofice-cold70%ethanolandcentrifugeagainfor30min. 7. Drythepelletsinspeec-vacfor10min. 8. ResUSPendDNAin100uLdH2Oovernight. 9. Transferin10uLaliquotsto384-wellplates(USAScientific#2802-0384orCorningCostar#6502)tomake10duplicateprintplatesets. 10. Drydownprintplatesetsinspeedvac. Tightlysealplateswithaluminumfoil(R.S.Hughes#425-3)forlong-termstorageatroomtemperature. 11. Beforeuse,resuspendoneprintsetin4uL3XSSCovernight. 12. SpotDNAontopolylysineslideswith16-tipor32-tiparrayer. Drydownusedprintplatesforstorageuntilnextuse.(Onesetofprintplatescanbeusedmultipletimes.)