AlanFung:LabNotes/CTCF/2011-5-10
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Bisulfite Conversion DNA Damage Estimation
- Perform PCR with CHR8, CHR21 and CHR22
- These will be our standards
File:ZhangLab 2 2011-05-10 10hr 40min.jpg
- Need to purify CHR8, CHR21, and CHR22.
- Then need to estimate the concentration
- Need amplicon length (~200)
- Run an experiment where we do PCR on both Bisulfite-converted DNA and the three controls
- Want to make sure number of starting molecules is the same.
- With normal DNA, 5 pg ~= 2 molecules
- With PCR product, X pg ~= 2 molecules; X << 5
- Need to run a PCR reaction using 500 pg of bisulfite converted DNA and X pg of PCR product as templates, with X such that the number of molecules in each tube is the same.
- Can run QPCR to check for DNA damage; only hot-start for 30 seconds instead of 3 minutes.