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