Kun:LabNotes/ExonomeSeq/2008-1-21
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Exp.1 Continue with the library construction from 01-18-08 experiment[edit]
Purify the end-repair reactions with MinElute columns[edit]
Ligation[edit]
N6 primed USER digested DNA 10ul 10ul 50uM Solexa_1 1ul 1ul 50uM Solexa_2 1ul 1ul 2X Quick ligation buffer 15ul 15ul Quick ligase 1ul 1ul H2O 2ul 2ul mix well -> room temperature for 20min -> 75C 10min Purified with Bioneer columns.
PAGE size selection[edit]
Nick-translation[edit]
DNA 15ul 10X ThermoPol Buffer 2ul 10mM dNTP 0.4ul 1mg/ml BSA 2ul 8U/ul Bst pol 1ul 65C 25min -> place on ice
PCR[edit]
Nick-translated DNA 5ul 2X iProof master mix 25ul 100uM Solexa_PCR_up 0.25ul 100uM Solexa_PCR_lo 0.25ul H2O 19.3ul 50X SYBG I 0.2ul 98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold
File:2008-01-22-E55kv1-SolLib-PCR.png