Alice:LabNotes/2008-7-17
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circularization of cosmic 3760 using CircleHelper[edit]
PCR[edit]
Step 1: Real time PCR reaction system X12 H2O 49.3ul 591.6ul SYBR green (50x) 0.4ul 4.8ul primer mix (100uM) 0.2ul 2.4ul Taq master mix(2x) 50ul 600ul probe template (cosmic 3760) 0.1ul 1.2ul Reaction program: 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> 15C hold
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.
Result: 16.1 ng/ul * 30ul = 483 ng
circularization[edit]
refer to LabNotes on [1] 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.