AlanFung:LabNotes/CTCF/2011-5-10

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Revision as of 23:32, 25 May 2011 by >Alan6017518 (→‎Prepare qPCR Master Mix)
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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.
    • 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

Perform real-time PCR reaction in thermocycler

      Step1   96C, 3m
      Step2   95C, 30s
      Step3   62C, 1m
      Step4   72C, 1m
      Step5   Go to step2 repeat 39 times
      Step6   72C, 5m
      Step7   4C,  Forever