Alice:LabNotes/2008-8-25
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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.