AlanFung:LabNotes/CTCF/2011-6-6

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Perform bisulfite conversion on 200ng of gDNA[edit]

Protocol[edit]

Setup PCR Reaction[edit]

  • Add the components on ice in order while mixing gently.
Component amount per reaction
distilled water 34
10x pfuturbo cx reaction buffer 5
dNTPs(10mM each DNTP) 1
DNA template 2
CHR8 F primer 3.3uM 3
CHR8 R primer 3.3um 3
sybr green 50x 1
pfuturbo cx hotstart dna polymerase 2.5U/ul 1
total reaction volume 50
  • Add 39ul master mix to primers and templates
    • 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[edit]

  • Amplification failed again.

CHR8 PCR amplification with iQ supermix[edit]

Objective[edit]

Confirm the bisulfite conversion works, iQ supermix has been the polymerase I used with the CHR primers and it works all the time. I will set up the reaction and confirm amplicons are successfully amplified before try with pfuturbo cx polymerase.

protocol[edit]

Component Amount per reaction
Distilled Water 18
gDNA Template (bisulfite converted gDNA GM12878 done on 6/6/2011) 1
CHR8 F primer 3.3uM 3
CHR8 R primer 3.3um 3
2x iQ Super Mix 25
Total reaction volume 50

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:060611 CHR8 iQ.jpg

  • Amplification is successful. Will run a gel after I repeat the amplification with pfu cx polymerase