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Kun:LabNotes/ExonomeSeq/2009-10-16
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===Batch 1=== *I did size selection on ~80ul of the sheared DNA from tube 1 using the SizeSelect gel, and extract ~100bp(L100) and ~120bp(L120) fragments. *I did End-repair on the extracted DNAs without further purification. DNA 45ul 10X End-repair buffer 5.3ul Enzyme Mix 2.5ul 25C for 45 min -> 70C for 10min -> purified with Qiaquick columns Ligation: Adaptor ligation: 20ul Solexa_1_up (200uM) + 20ul Solexa_1_lo_noP (200uM) + 5ul 10X Stoffel buffer + 55ul H2O -> 94C 3min -> -0.1C/sec to 20C DNA 30ul 40uM adaptors 3ul 2x QuickLigase Buffer 34ul QuickLigase 1ul RT 15min -> purified with Qiaquick columns -> extract 120bp+ and 150bp+ fragments with SizeSelect gel. PCR F+R1 F+R2 F Template 5ul 5ul 5ul 2x iProof Mix 50ul 50ul 50ul Solexa_PCR_up(10uM) 2ul 2ul 2ul AmpR6.3Sol(100uM) 0.4ul - - AmpF6.3rSol(100uM) - 0.4ul - 50X SYBGI 0.4ul 0.4ul 0.4ul H2O 44ul 44ul 44ul 98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 10sec) x 8 -> (98C 10sec -> 72C 15sec) x 10 -> 72C 2min -> 15C hold.
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