AlanFung:LabNotes/CTCF/2011-6-7: Difference between revisions
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==Perform bisulfite conversion on 6ng, 1.2ng, and 600pg of gDNA using Zymo’s direct kit== | ==Perform bisulfite conversion on 6ng, 1.2ng, and 600pg of gDNA using Zymo’s direct kit== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Tube''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount''' | | align="center" style="background:#f0f0f0;"|'''Amount''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume per Reaction''' | | align="center" style="background:#f0f0f0;"|'''Volume per Reaction''' | ||
Line 42: | Line 43: | ||
| align="center" style="background:#f0f0f0;"|'''H2O''' | | align="center" style="background:#f0f0f0;"|'''H2O''' | ||
|- | |- | ||
| 6ng||1.2||5ng/ul||18.8 | | A||6ng||1.2||5ng/ul||18.8 | ||
|- | |- | ||
| 1.2ng||6.82||176g/ul||13.18 | | B||1.2ng||6.82||176g/ul||13.18 | ||
|- | |- | ||
| 600pg||3.41||176g/ul||16.59 | | 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 | *Add 130ul CT Conversion Reagent to each tube | ||
==Synthesize 2nd strand with N9 primer and klenow exo== | ==Synthesize 2nd strand with N9 primer and klenow exo== | ||
==Perform standard tagmentation and PCR amplification with orange/blue primers== | ==Perform standard tagmentation and PCR amplification with orange/blue primers== |
Revision as of 21:45, 7 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
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