AlanFung:LabNotes/CTCF/2011-6-6: Difference between revisions
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{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''amount per reaction''' | ||
|- | |- | ||
| | | distilled water||34 | ||
|- | |- | ||
| | | 10x pfuturbo cx reaction buffer||5 | ||
|- | |- | ||
| dNTPs (10mM each DNTP)||1 | | dNTPs(10mM each DNTP)||1 | ||
|- | |- | ||
| DNA template | | DNA template||2 | ||
|- | |- | ||
| CHR8 F | | CHR8 F primer 3.3uM||3 | ||
|- | |- | ||
| CHR8 R | | CHR8 R primer 3.3um||3 | ||
|- | |- | ||
| | | sybr green 50x||1 | ||
|- | |- | ||
| | | pfuturbo cx hotstart dna polymerase 2.5U/ul||1 | ||
|- | |- | ||
| | | total reaction volume||50 | ||
|} | |} | ||
*Add 39ul master mix to primers and templates | *Add 39ul master mix to primers and templates |
Revision as of 20:52, 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 |
distilled water | 34 |
10x pfuturbo cx reaction buffer | 5 |
dNTPs(10mM each DNTP) | 1 |
DNA template | 2 |
CHR8 F primer 3.3uM | 3 |
CHR8 R primer 3.3um | 3 |
sybr green 50x | 1 |
pfuturbo cx hotstart dna polymerase 2.5U/ul | 1 |
total reaction volume | 50 |
- Add 39ul master mix to primers and templates
- 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