Jie:LabNotes/ExonomeSeq/2008-7-17
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circularization of cosmic 3760[edit]
PCR[edit]
T7 exonuclease digestion[edit]
add 2ul T7 exonuclease (10u/ul) in each tube. 25C 1h. Take 4ul to run the TBU gel. Purify with Qiaquick column. Elute in 30ul ddH2O. Measure with nanodrop.
circularization[edit]
template 30ul 10x Ampligase Buffer 5ul Circlehelper(10uM) 0.25ul dNTP(10mM) 0.5ul Stoffel fragment( ) 0.5ul Ampligase 1ul ddH2O ul 94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C .
check product on TBU gel and do PAGE purification of digested product.