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