Kun:LabNotes/ExonomeSeq/2008-1-8

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Exp.1 Perform 2nd round PCR on the PAGE purified 450bp band from the previous capturing experiment.

                         dNTP       dUTP
    Template:            5ul       5ul
    10X PCR buffer:     10ul      10ul
    10mM dNTP:           2ul       2ul
    1mM dUTP:            0ul       1ul
    100uM AmpFSolV6.2  0.4ul     0.4ul
    100uM AmpRSolV6.2  0.4ul     0.4ul
    50X SYBG I:        0.4ul     0.4ul
    JumpStart Taq:       2ul       2ul
    H2O:                80ul      79ul

94C 3min -> (94C 30sec -> 72C 1min) x 10cyc -> 72C 3min -> 15C hold. Purify 50ul with Bioneer columns. dNTP: 31ng/ul x 30ul dUTP: 30ng/ul x 30ul

Exp.2 Make sequencing library by USER digestion and ligation

15ul dUTP amplicon -> add 1.5ul USER enzyme -> 37C 1h -> set up ligation.

Exp. 3 Make sequencing library by random priming

                       SolF/Rn6     N6
     DNA                  5ul       5ul
     10X Klenow buffer  2.5ul     2.5ul
     100uM SolFn6      0.25ul       0ul
     100uM SolRn6      0.25ul       0ul
     1mM N6               0ul     0.5ul
     10mM dNTP            1ul       1ul
     H2O                 14ul      14ul

94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> purified with MinElute columns.

Setup ligation for the N6 reaction.

Exp.2 Perform another rounf of exon capture experiment

   Sheared NA10835(~300ng/ul)                3ul
   E55kv1 probes 107nM (01/07/08,purified)  17ul
   10X Ampligase buffer                   2.25ul

95C 10min -> 60C 40h -> add 2.5ul NSL mix -> 60C 1h -> (95C 2min -> 60C 1h) x 20 -> 37C 1min -> add 2ul exo I&III, 1ul T7 exo -> 37C 2h -> 95C 2min -> 4c hold