AlanFung:LabNotes/CTCF/2011-6-7: Difference between revisions

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>Alan6017518
>Alan6017518
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| 600pg||3.41||176g/ul||16.59
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*Add 130ul CT Conversion Reagent to each tube

Revision as of 21:04, 7 June 2011

qPCR with CHR8 primer using pfu Cx polymerase on bisulfite converted GM12878 gDNA

Protocol

Component amount per reaction
distilled water 35.6
10x pfuturbo cx reaction buffer 5
dNTPs(10mM each DNTP) 1
DNA template 1
CHR8 F primer 3.3uM 3
CHR8 R primer 3.3um 3
sybr green 50x 0.4
pfuturbo cx hotstart dna polymerase 2.5U/ul 1
total reaction volume 50
    • Perform Manufacturer protocol for PfuTurbo Cx Hotstart DNA Polymerase
95C 2m
95C 30s
52C 30s
72C 2m
Plate Read
Go to step 2 repeat for 29 times
72C 10m
15C hold

Results

File:060611 CHR8 pfuCx.jpg

  • Amplification failed with Pfu cx polymerase

Perform bisulfite conversion on 6ng, 1.2ng, and 600pg of gDNA using Zymo’s direct kit

Amount Volume per Reaction Tube Concentration H2O
6ng 1.2 5ng/ul 18.8
1.2ng 6.82 176g/ul 13.18
600pg 3.41 176g/ul 16.59
  • Add 130ul CT Conversion Reagent to each tube