AlanFung:LabNotes/CTCF/2011-8-10

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Objective[edit]

  • Athurva's sequencing results came back, most of them failed and the one that worked reveals that the library is not bisulfite converted
  • We will run a validation PCR to confirm the methylation status with the 3 set of primers CHR8,21 and 22

Protocol[edit]

  • Prepare Master Mix for the 3 sets of primers
Content Volume per reaction MM
ddH2O 7 29.4
Primer F (3.3uM) 6 25.2
Primer R (3.3uM) 6 25.2
gDNA 1 Do not add to MM
2X iQ SuperMix 20 84
Total 40 163.8
  • Plate Layout
  • Add 1ul of DNA to wells and add 39ul MM to each well accordingly
Chr8 Chr21 Chr22
Negative Negative Negative
150 150 150
1500 1500 1500
Positive Positive Positive

Perform 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

Results[edit]

File:ZhangLab 2 2011-08-10 17hr 01min.jpg

  • It didn't look like the bisulfite conversion worked, no bands around 200bp for chr8 and 22 on sample 150 cells and 1500 cells, cannot draw conclusion from CHR21 for it has a lot of unspecific bands
  • However Athurva has some promising results from sanger sequencing of his libraries, it appears that the sample was bisulfite converted.