AlanFung:LabNotes/CTCF/2011-6-6: Difference between revisions
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==Perform bisulfite conversion on 200ng of gDNA== | ==Perform bisulfite conversion on 200ng of gDNA== | ||
==Protocol== | |||
===Setup PCR Reaction=== | |||
*Add the components on ice in order while mixing gently. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount per reaction''' | |||
| align="center" style="background:#f0f0f0;"|'''x6.6''' | |||
|- | |||
| Distilled water||31||204.6 | |||
|- | |||
| 10X pfuTurbo Cx reaction buffer||5||33 | |||
|- | |||
| dNTPs (10mM each DNTP)||1||6.6 | |||
|- | |||
| DNA template/STD||5|| | |||
|- | |||
| CHR8 F Primer (3.3uM)||3|| | |||
|- | |||
| CHR8 R Primer (3.3uM)||3|| | |||
|- | |||
| SYBR Green (50X)||1||6.6 | |||
|- | |||
| PfuTurbo Cx Hotstart DNA Polymerase (2.5U/ul)||1||6.6 | |||
|- | |||
| Total reaction volume||50||257.4 | |||
|} | |||
*Add 39ul master mix to primers and templates | |||
**Perform Manufacturer protocol for PfuTurbo Cx Hotstart DNA Polymerase | |||
95C 2m | |||
95C 30s | |||
58C 30s | |||
72C 1m | |||
Plate Read | |||
Go to step 2 repeat for 29 times | |||
72C 10m | |||
15C hold | |||
==Results== |
Revision as of 20:37, 6 June 2011
Perform bisulfite conversion on 200ng of gDNA
Protocol
Setup PCR Reaction
- Add the components on ice in order while mixing gently.
Component | Amount per reaction | x6.6 |
Distilled water | 31 | 204.6 |
10X pfuTurbo Cx reaction buffer | 5 | 33 |
dNTPs (10mM each DNTP) | 1 | 6.6 |
DNA template/STD | 5 | |
CHR8 F Primer (3.3uM) | 3 | |
CHR8 R Primer (3.3uM) | 3 | |
SYBR Green (50X) | 1 | 6.6 |
PfuTurbo Cx Hotstart DNA Polymerase (2.5U/ul) | 1 | 6.6 |
Total reaction volume | 50 | 257.4 |
- Add 39ul master mix to primers and templates
- Perform Manufacturer protocol for PfuTurbo Cx Hotstart DNA Polymerase
95C 2m 95C 30s 58C 30s 72C 1m Plate Read Go to step 2 repeat for 29 times 72C 10m 15C hold