Revision as of 23:49, 7 June 2011 by >Alan6017518
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
Tube
|
Amount
|
Volume per Reaction
|
Tube Concentration
|
H2O
|
A |
6ng |
1.2 |
5ng/ul |
18.8
|
B |
1.2ng |
6.82 |
176g/ul |
13.18
|
C |
600pg |
3.41 |
176g/ul |
16.59
|
D |
Blank |
0 |
N/A |
20
|
- Warm CT conversion Reagent (prepared on 6/6/11) up to 37C and vortex in 37C incubator
- Add 130ul CT Conversion Reagent to each tube
Synthesize 2nd strand with N9 primer and klenow exo
- Add 1 uL Klenow Polymerase(1U/ul) and
- Incubate for 10min at 30 C
Perform standard tagmentation and PCR amplification with orange/blue primers