AlanFung:LabNotes/CTCF/2011-5-10: Difference between revisions

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*CHR21-4.39ng/ul 36.09nM
*CHR21-4.39ng/ul 36.09nM
*CHR22-4.77ng/ul 39.21nM
*CHR22-4.77ng/ul 39.21nM
* 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.
===Determine the efficiency of bisulfite conversion===
===Determine the efficiency of bisulfite conversion===
*After performing the bisulfite conversion procedures, we should perform real-time PCR of the samples and controls to determine the amount of unconverted gDNA vs. converted human genomic DNA. Base on the difference in the CT values, we can determine the conversion efficiency.
*After performing the bisulfite conversion procedures, we should perform real-time PCR of the samples and controls to determine the amount of unconverted gDNA vs. converted human genomic DNA. Base on the difference in the CT values, we can determine the conversion efficiency.

Revision as of 21:50, 25 May 2011

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.
    • Qubit Results(ng/ul)
    • Assume amplicon length ~200bp
  • CHR8-3.25ng/ul 26.72nM
  • CHR21-4.39ng/ul 36.09nM
  • CHR22-4.77ng/ul 39.21nM

Determine the efficiency of bisulfite conversion

  • After performing the bisulfite conversion procedures, we should perform real-time PCR of the samples and controls to determine the amount of unconverted gDNA vs. converted human genomic DNA. Base on the difference in the CT values, we can determine the conversion efficiency.