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