Kun:LabNotes/CpgSeq/2007-12-12

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Exp.1 Colony PCR[edit]

I plated 25ul of the transformation from RL1 and RL2 yesterday, and got about 100 colonies per plate today. I decided to set up PCR for 16 colonies per plate and send out some amplicons for sequencing.

    2X Taq Matermix    25ul
    100uM M13F        0.1ul
    100uM M13R        0.1ul
    H2O                25ul

94C 3min -> (94C 30sec -> 55C 30sec -> 72C 1min) x 35 -> 72C 3min -> 4C hold.

 File:2007-12-13-colony-PCR.png
 File:2007-12-13-colony-PCR-PAGE.png

I got the Sanger sequencing result back for RL1a, RL1b, RL2a and RL2b. None of the inserts has homology to any human sequences. RL1a is closest to Caulobacter??? The GC content of the sequences are not low, suggesting that they are not from bisulfite converted DNA. It's unclear to me what these sequences are.