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.