SREBF1 Genotyping Protocol
Revision as of 16:32, 30 August 2016 by Reddj (Talk | contribs) (Created page with "==Primers== *Srebf1 WT Fwd: 5' CCA TGT GCG CTC ACC CGA G 3' *Srebf1 All Fwd: 5' CGA TCC GCT GTA GAG ACC CT 3' *Srebf1 All REV: 5' CAT TCA GAG CAC CCG GTG AA 3' ==Primer...")
Primers
- Srebf1 WT Fwd: 5' CCA TGT GCG CTC ACC CGA G 3'
- Srebf1 All Fwd: 5' CGA TCC GCT GTA GAG ACC CT 3'
- Srebf1 All REV: 5' CAT TCA GAG CAC CCG GTG AA 3'
Primer Mixes
Primer mixes are resuspended at 100uM by adding 10 times the nmol amount on the tube. (For example: nmol=32.4, add 324 uL of water to primer tube).
Add 2uL of each primer (WT Fwd, All Fwd, and All Rev) to 494uL of ddH2O to make a total volume of 500uL of diluted primer.
PCR Reaction
The PCR program is called "srebp" on the thermal cycler and is as follows:
- 95°C 10 min
- 95°C 10 s
- 57.5°C 30 s
- 72°C 45 s
- Go to step 2, 34 times (total of 35 cycles)
- 72°C 5 min
Bands
For mutants 1 and 2 (m1 and m2) the WT bands are the same: 302 and 218 For m1: the mutant band is at 277 (heterozygotes should have bands at 302, 218, and 277) For m2: the mutant band is at 150 (heterozygotes should have bands at 302, 218, and 150)