SREBF1 Genotyping Protocol

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)