AlanFung:LabNotes/CTCF/2011-6-6
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Perform bisulfite conversion on 200ng of gDNA
Protocol
Setup PCR Reaction
- 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
- Amplification failed again.
CHR8 PCR amplification with iQ supermix
Objective
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
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