AlanFung:LabNotes/CTCF/2011-6-7: Difference between revisions
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==Perform standard tagmentation and PCR amplification with orange/blue primers using KAPA== | ==Perform standard tagmentation and PCR amplification with orange/blue primers using KAPA== | ||
*Scale up volume accordingly (~12ul) | *Scale up volume accordingly (total volume ~12ul after 2nd strand synthesis) | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Content''' | | align="center" style="background:#f0f0f0;"|'''Content''' |
Revision as of 17:28, 9 June 2011
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
- 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 0.52ul 5uM N9 Primers
- 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 (total volume ~12ul after 2nd strand synthesis)
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