Alice:LabNotes/2008-8-25

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Revision as of 17:09, 25 August 2008 by >Jie deng
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circularization of cpg probe using CircleHelper

PCR reaction system                                       X48              
H2O                                       50ul          2400ul              
SYBR  green (50x)                        0.4ul          19.2ul
primer mix (100uM)                       0.2ul          9.6ul                 
Taq master mix(2x)                        50ul          2400ul 
probe template (cpg30K V2.3)             0.05ul         2.4ul

94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min ->
15C hold

cpg template is used instead since cosmic template is used up. and it is used only to check the accuracy of the protocol.

T7 exonuclease digestion

Purify the remaining 40 tubes using ethanol purification. elute in 200ul of ddH2O. Add ratio if 0.5ul/tube amount of T7
exonuclease into the product and 10X NEBuffer#4. 25C 1h. Purify with Qiaquick column. Elute in 30ul ddH2O. Measure with
nanodrop. 

Result: 

circularization reaction

template                          ul
10x Ampligase Buffer              ul  
CircleHelper V4.2(10uM)            ul  reactio system 2 uses CircleHelper V4.2.2  0.6ul
dNTP(10mM)                      0.5ul
Stoffel fragment(10U/ul)        0.5ul
Ampligase                         1ul
ddH2O                              ul

94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C .

check product on TBU gel and column purification of digested product.
Gel image after circlehelper insertion:

Annealing with AluI oligo and digestion with ALuI

1. Add 1ul NEB buffer2, 1 uL of 100 uM RE-AluI-V6_2_Ida guide oligo in 5ul circularized probe from previous steps.
2. Denature at 95C in PCR machine for 10 mins. Turn off the machine and wait for 20 min.
3. Add 2uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours.