AlanFung:LabNotes/CTCF/2011-9-7

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Bisulflash Kit Test[edit]

  • Athurva and I will test the bisulflash kit from Epigentek
  • We will perform bisulfite conversion on 100, 10 & 1 ng of Jurkat gDNA with NTC
  • We will verify the completeness of bisulfite conversion based on quantitative qPCR and MDA

Bisulfite Conversion[edit]

  • Done by Athurva
  • Elute each samples with 15ul of elution buffer and we will use 5ul for qPCR, 5ul for MDA and 2 ul for a gel quantification

qPCR Analysis with Primer set CHR22[edit]

  • We picked CHR 22 set, since it has the smallest amplicon size among the 3 sets of primers we have, bisulfite conversion by bisulFlash kit produces ~200bp fragments, chances of primer sites being separated is very possible.
Content Volume per Reaction MM qPCR Setup
2X iQ Supermix 20 88 NTC
Primer 22F 6 26.4 1ng
Primer 22R 6 26.4 10ng
gDNA 5 Do not add to MM 100ng
H2O 3 13.2
Total 40 154
  • Add 35ul MM to each samples
     Step1   96C, 3m
     Step2   95C, 30s
     Step3   62C, 1m
     Step4   72C, 1m
     Step5   Go to step2 repeat 39 times
     Step6   72C, 5m
     Step7   15C,  Forever

MDA Analysis[edit]

  • Denature all samples at 95C for 30sec and quickly transfer to cooler rack
Content Volume per Reaction MM
10X Repli Phi Buffer 2 8.8
1mM N9 Primer 1 4.4
25mM dNTP 0.8 3.52
Repliphi phi 29 (100U/ul) 1 4.4
Klenow Exo Minus (10U/ul) 0.5 2.2
50X SYBG 0.1 0.44
H2O 9.6 42.24
Modified DNA Sample 5 Do not add to MM
Total 20 66
  • Add 15ul of MDA mastermix to each sample
  • 30C 120 min, 65C 10 min, 15C hold

PAGE Gel Analysis[edit]

File:ZhangLab 2 2011-09-08 10hr 40min.jpg

Results[edit]

The bisulfite conversion is successful, verfied by the PCR validation test. Yet sample loss is a huge issue for this kit, we dont obtain expected yield from the bisuflash kit.