Kun:LabNotes/CpgSeq/2007-11-1

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Exp. 1 Bisulfite conversion of genomic DNA with EpiTect kit[edit]

Genomic DNA samples (conc. measured by Nanodrop):

  • DNA RL (from Dr. Huidong Shi), 185ng/ul
  • Jurkat gDNA (from NEB), 194ng/ul

Resuspend one vial of Bisulfite Mix in 800ul nuclease free water, votex for 5min. Set up reactions:

                                  RL          Jurkat
    DNA                          5ul x 2       5ul x 2
    Water                       15ul x 2      15ul x 2
    Bisulfite mix               85ul x 2      85ul x 2
    DNA Protection Buffer       35ul x 2      35ul x 2

Perform the bisulfite conversion on PTC-200 using the EpiTect program 99C5min->60C20min->99C5min->60C1h25min->99C5min->60C2h55min->20Chold.

Follow the EpiTect protocol to clean up the bisulphite converted DNAs. Check with argarose gel.

I measured the DNA concentration with Nanodrop. They are unusually high (>1ug/ul), and the OD260/280 ratio is also too high ~3.4. I'm wondering whether it is due to precipitants in the buffer BD. Need to give Qiagen's tech support a call.

    File:2007-11-02-bisulfite-conversion.png