qPCR with CHR8 primer using pfu Cx polymerase on bisulfite converted GM12878 gDNA[edit]
Protocol[edit]
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[edit]
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[edit]
Objective[edit]
- test the feasibility of using zymo direct kit to perform bisulfite conversion on 100 cells, 200 cells and 1000 cells worth of DNA and perform 2nd strand synthesis to make sequencing library with regular nextera kit
- Test wether we need to perform heat denaturing on the bisulfite converted library to ensure we have single stranded DNA after conversion with 3 set of samples.
Protocol[edit]
- 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 10ul elution buffer
- Transfer to qPCR tubes
Content
|
Volume per reaction
|
X13.2
|
50uM N9 Primer |
0.6 |
7.92
|
dNTPs (10mM each DNTP) |
1 |
13.2
|
5X Nextera LMW Buffer |
3 |
39.6
|
Total |
4.6 |
|
- Add 4.6ul to each reaction
Synthesize 2nd strand with N9 primer and klenow exo[edit]
- Set 1 denature at 95C
- Set 2 denature at 84C
- Set 3 no heat denaturing
- Heat up to 95C, 84C and no heating for 2 mins and quickly transfer to cooling rack, wait for 1 min.
- Add 0.5 uL Klenow Exo-(10U/ul)
- Incubate at 25C for 10min
- Incubate at 30C for 20min
Perform standard tagmentation and PCR amplification with orange/blue primers using KAPA[edit]
- Add 1ul 1:50 Nextera illumina compatible transposomes to set 1A, 2A & 3A
- Make 1:10 Nextera illumina compatible transposome (2 μl Nextera enzyme, 8 μl TE, 10 μl 100% gly- cerol)
- Add 1ul 1:10 Nextera illumina compatible transposomes to set 1B, 2B, 3B, 1C, 2C &3C
- Incubate at 55C for 5min
- Mix on ice
Content
|
Volume per reaction
|
X13.2
|
Nuclease Free Water |
8 |
105.6
|
10uM Orange Primer |
1 |
13.2
|
10uM Blue Primer |
1 |
13.2
|
Bisulfite converted DNA w/ 2nd strand |
15 |
|
Kapa SYBR Green Supermix |
25 |
330
|
Total |
50 |
660
|
- Add 35ul master mix to each well
- Perform Manufacturer protocol for KAPA
95C 3m
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 24
72C 10m
Hold 15C
- Cycle the samples in a thermocycler under the following conditions
- 3. Purify the tagged DNA using qiaquick column
- Page Quantification
Results[edit]
File:ZhangLab 2 2011-06-10 13hr 13min.jpg
File:ZhangLab 2 2011-06-10 13hr 15min.jpg
- Very dim bands on the libraries
- qPCR curve ramps up very early (after 1x cycles)
- Primer dimer issue?