Revision as of 17:23, 9 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
- Setup 3 sets of reactions (Set 1, 2 & 3)
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
- Elute with 11ul elution buffer
Synthesize 2nd strand with N9 primer and klenow exo
- Set 1 denature at 95C
- Set 2 denature at 84C
- Set 3 no heat denaturing
- Heat up to 95C, 84C and no heating for 7 mins and quickly transfer to cooling rack, wait for 1 min.
- Add 1 uL Klenow Polymerase(1U/ul)
- Add N9 Primers @ 0.2uM
- Add 1ul dNTPs (10mM each DNTP)
- Incubate for 10min at 30 C
Perform standard tagmentation and PCR amplification with orange/blue primers using KAPA
- Scale up volume accordingly
Content
|
Volume per reaction
|
gDNA |
3
|
1:50 Nextera illumina compatible transposomes |
1
|
5X Nextera HMW Buffer |
1
|
- Incubate at 55C for 5min
- Mix on ice
Content
|
Volume per reaction
|
Nuclease Free Water |
17.6
|
10uM Orange Primer |
1
|
10uM Blue Primer |
1
|
Bisulfite converted DNA |
5
|
50X SYBR Green |
0.4
|
Kapa Supermix |
25
|
Total |
50
|
- Perform Manufacturer protocol for KAPA
95C 3m
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 34
72C 10m
Hold 15C
- Cycle the samples in a thermocycler under the following conditions
- 3. Purify the tagged DNA using qiaquick column
- Page Quantification