Jie:LabNotes/CpgSeq/2009-9-28
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Exom-samples Sequencing library construction without shearing
PCR with Amp6.3NH2 primer
I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample. reaction system x4 x6 H2O 45ul 180ul 1080 2x iProof Mastermix 50ul 200ul 1200 AmpF6.3NH2(10uM) 2ul 8ul 48 AmpR6.3NH2(10uM) 2ul 8ul 48 50x SYBG I 0.4ul 1.6ul 9.6 template 1ul/each for purified amplicons Total 100ul 800ul 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->72C 3min. Purify with Qiaquick column. Elute in 100ul EB.
MmeI digestion
Digestion with MmeI x7 Total 50ul PCR 28ul 10X NEBuffer 4 5ul 35 1mM SAM(fresh) 10ul 70 2U/ul Mme I 8ul 56
1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. Qiaquick column purification. Elute in 30ul EB.