AlanFung:LabNotes/CTCF/2011-5-10: Difference between revisions
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==Prepare qPCR Master Mix== | ==Prepare qPCR Master Mix== | ||
*Add 1ml of Illumina GA Primer Premix(10X) to 5ml bottle of KAPA SYBR FAST qPCR Master Mix (2X) and mix well | *Add 1ml of Illumina GA Primer Premix(10X) to 5ml bottle of KAPA SYBR FAST qPCR Master Mix (2X) and mix well | ||
{| {{table}} | |||
| ||CHR8||CHR21||CHR22 | |||
|- | |||
| 2X iQ Super Mix||20||20||20 | |||
|- | |||
| Primer F+R (1.65uM)||4.85||4.85||4.85 | |||
|- | |||
| gDNA||8||8||8 | |||
|- | |||
| ddH2O||7.15||7.15||7.15 | |||
|- | |||
| Total||40||40||40 | |||
|} |
Revision as of 23:28, 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.
Make Serial Dilution on Control DNA
- Create 6 STDs
Sample Name | dsDNA Concentration (pM) |
Std 1 | 20 |
Std 2 | 2 |
Std 3 | 0.2 |
Std 4 | 0.02 |
Std 5 | 0.002 |
Std 6 | 0.0002 |
Prepare qPCR Reagents
Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing
- 2X KAPA SYBR FAST qPCR Master Mix
- 6 DNA Standards
- 1:1000 dilution of bisulfite converted library DNA
- Primer Mix (F+R) 1:1 ratio 1.65uM
Prepare qPCR Master Mix
- Add 1ml of Illumina GA Primer Premix(10X) to 5ml bottle of KAPA SYBR FAST qPCR Master Mix (2X) and mix well
CHR8 | CHR21 | CHR22 | |
2X iQ Super Mix | 20 | 20 | 20 |
Primer F+R (1.65uM) | 4.85 | 4.85 | 4.85 |
gDNA | 8 | 8 | 8 |
ddH2O | 7.15 | 7.15 | 7.15 |
Total | 40 | 40 | 40 |