Alice:LabNotes/2008-8-25
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circularization of cpg probe using CircleHelper[edit]
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[edit]
*first 8 tubes were used last week to check the accuracy of the protocol* 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: tube 1: 92.9ng/ul*30ul tube 2: 105.5ng/ul*30ul
treat amplified probe with OptiKinase[edit]
Since the primer Ap1V4IU was not phosphorelated at 5 terminal, the amplified probe need to be phosphorelated so that the Ampligase could work. x3 cpg30KV2.2(after Exonuclease, 144ng/ul) 10ul(30nmol) 30ul Optikinase reaction buffer 10x 4ul 12 ATP(10mM) 4ul 12 H2O 16ul 48 Optikinase 6ul 18 37C 30mins -> 65C 10mins ->4C. Quick purification. Elute in 30ul EB. NanoDrop result: 67.8 ng/ul/(144x330)= 1.43 uM 71.5 ng/ul/(144*330)= 1.50 uM So 1 ul of probe need 0.14 ul of CircleHelper(10uM)
circularization reaction[edit]
template 5ul 10x Ampligase Buffer 2ul CircleHelper V4.2(10uM) 0.7ul reaction system 2 uses Circle Helper V4.2.2(10uM) dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase 1ul ddH2O 11.3ul 94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C . check product on TBU gel and gel purification of digested product. Gel image after circlehelper insertion: File:8-27-08 insert circlehelper after optikinase.jpg Result: since the gel shows multiple bands, so we are not certain which band is the one we need. Therefore prior to gel purification, it needs to be treated with exonuclease to get rid of any linear DNA. Add 2ul of Exonuclease I&III, 37C 2hrs -> 95C 10min -> 4C
Annealing with AluI oligo and digestion with ALuI[edit]
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.