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Kun:LabNotes/CpgSeq/2007-12-10
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==Exp. 1 PAGE purification on the CpG MIP PCR amplicons did on 12/09/2007== I hope that one of the 300- or 400+ bands are from the captured CpG sequences. To confirm this, I will do PAGE purification, then re-amplify, PCR cloning and sequence a few clones. [[Image:2007-12-10-CpGMIP-PCR-PAGE-purification.png|300px]] #After elution of DNA from sheared gel with 100ul 1X TE, perform PCR (without ethanol precipitation). I included the Hd48MIP1, 2, 5 primers in addition to the AmpF/RSol. Each 4x4=16 Template: 1ul 5x Phusion buffer: 5ul 80ul 10mM dNTP: 0.5ul 8ul 10uM Primers: 1.25ul 5ul x 4 Phusion pol: 0.25ul 4ul 50X SYBG I: 0.2ul 3.2ul H2O: 16.8ul 272ul 98C 30sec -> 20 cycles of (98C 10sec -> 59C (or 72C) 20 sec -> 72C 20sec) -> 72C 5min [[Image:2007-12-10-CpG-MIP-PCR-PAGE-PCR2.png|500px]] I paused the thermocycling after the 16th cycle, and took the amplicons with AmpF/RSol out for cloning. Ligation: pCR-Blunt 0.5ul Blunt PCR product 2ul 10X Ligation buffer 0.5ul Sterile water 1.5ul T4 DNA ligase (4U/ul) 0.5ul 16C 1h -> 65C 10min -> 4C hold. From the PCR result, it doesn't look like the PAGE purified fragments contain any target sequence. But I could be wrong since the PCR condition is different. I decided to do transfection on the RL1 and RL2 ligation reactions. ==Exp. 2 Confirmation of CpG MIP capturing experiment by PCR== Another way to confirm the result of the MIP capturing experiment is to perform PCR on the circularization reactions with target-specific PCR primers. I will use the bisulfite converted DNA as positive controls. The primers used included HD48MIP 1-3. Template: 1ul (1:100 dilution of bifulfite converted gDNAs) 10X buffer: 2.5ul 10mM dNTP: 0.5ul 10uM Primer pair 0.5ul 50X SYBR I 0.2ul JumpStart Taq 0.25ul H2O 20ul Total number of reactions: 4 MIP reactions x 3 primer pairs + 2 control x 3 primer pairs = 18 94C 3min -> 30 cycles of (94C 30min -> 57C 45sec -> 72C 30sec) -> 72C 3min [[Image:2007-12-10-CpGMIP-PCR-PAGE-PCR.png|500px]] The results are noisy. The amplicons with Hd48MIP2 sort of suggest that there might be some targeted DNA being captured, but there are too many non-specific bands. Hd48MIP1&5 didn't even work at all. I will repeat this PCR using a touch-down protocol.
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