Jie:LabNotes/CpgSeq/2008-8-22

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Treat circlehelper with Antarctic phosphatase 2008-08-21[edit]

 circlehelper_V4.2_2_Ida (100uM)      2ul
 10x Antarctic phosphatase buffer     1ul
 Antarctic phosphatase (5U/ul)        2ul
 ddH2O                                5ul
 37C 25mins -> 90C 5mins.


annealing of cirlehelperV4.2_2_Ida (2008-08-22)[edit]

The probe:circlehelper is expected to be 1:1. 
Probe concentration is: 144 ng/ul /(144x330) = 303 nM. 6ul of probe need CircleHelper(100uM) 0.2ul.
Annealing system is setup is as follows:                  
                                tube1   x  3       tube2 x3                  tube2     x3
template                         6ul       18       same as tube1              6ul      18 
10x Ampligase Buffer             1ul        3     except that                  1ul       3
Circlehelper_V4.2(100uM)       0.2ul      0.6     circlehelp_V4.2_2_Ida        1ul       3(phosphotase treated V4.2_2_Ida)
dNTP(10mM)                     0.5ul      1.5                                  0.5ul     1.5
Stoffel fragment(10U/ul)       0.5ul      1.5                                  0.5ul     1.5
Ampligase                        1ul        3                                    1ul       3
 
94C 3min -> 5 cycles X (94C 1 min -> 60C 10mins) -> 95C 1min ->add ExoI/III 2ul -> 37C 1h -> 95C 5mins -> 4C.
 PAGE cut and ethanol precipitation.

amplification of selected size with AmpV6_2F/R and AP1V4IU/Ap2V4[edit]

File:20080824 amplification of selected size with AmpV6 2F R and AP1V4IU Ap2V4.jpgamplification of selected size with AmpV6.2F/R and AP1V4IU/Ap2V4

annealing with AluI oligo and digestion with ALuI[edit]

1. Add 1ul NEB buffer2, 3 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.
2. Add 2uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours.

File:20080823 after AluI digestion.jpg20080823_after AluI digestion