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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)
[[Category: Genotyping]]
[[Category: Mouse Work]]
[[ Category:PCR]]
[[ Category:DNA]]
[[ Category:Molecular Biology]]
*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)
[[Category: Genotyping]]
[[Category: Mouse Work]]
[[ Category:PCR]]
[[ Category:DNA]]
[[ Category:Molecular Biology]]