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.
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