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[edit]

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.

 File:2007-12-10-CpGMIP-PCR-PAGE-purification.png
  1. 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

 File:2007-12-10-CpG-MIP-PCR-PAGE-PCR2.png

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[edit]

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

 File:2007-12-10-CpGMIP-PCR-PAGE-PCR.png

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.