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.