Alice:LabNotes/2008-9-1

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Experiment: Extending the cosmic probe length[edit]

previous steps and the over-view of the experiment can be found through this link [[1]]

circularization reaction[edit]

template                             2ul
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                             14.3ul

94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C .
TBU gel after the circularization reaction:
File:9-1-08.jpg

check product on TBU gel and MinElute purification of digested product.

Annealing with AluI oligo and digestion with ALuI[edit]

1. Add 2ul NEB buffer2, 2uL of 100 uM RE-AluI-V6_2_Ida guide oligo in 10ul 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 4uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours.
File:ZhangLab 2 2008-09-02 16hr 52min.jpg