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.