AlanFung:LabNotes/CTCF/2011-5-10: Difference between revisions
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[[File:ZhangLab_2 2011-05-10 10hr 40min.jpg|500px]] | [[File:ZhangLab_2 2011-05-10 10hr 40min.jpg|500px]] | ||
* Need to purify CHR8, CHR21, and CHR22. | * Need to purify CHR8, CHR21, and CHR22. | ||
**Qubit Results | **Qubit Results(ng/ul) | ||
**Assume amplicon length ~200bp | |||
*CHR8-3.25ng/ul 26.72nM | *CHR8-3.25ng/ul 26.72nM | ||
*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 | * 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. | ** Want to make sure number of starting molecules is the same. |
Revision as of 21:40, 24 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
- 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.