SREBF1 Genotyping Protocol - Revision history

Reddj at 18:27, 30 August 2016

2016-08-30T18:27:36Z

← Older revision		Revision as of 18:27, 30 August 2016
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	For mutants 1 and 2 (m1 and m2) the WT bands are the same: 302 and 218		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 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)		For m2: the mutant band is at 150 (heterozygotes should have bands at 302, 218, and 150)

Reddj at 18:26, 30 August 2016

2016-08-30T18:26:55Z

← Older revision		Revision as of 18:26, 30 August 2016
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	[[File:Screen Shot 2016-08-30 at 12.48.27 PM.png]]		[[File:Screen Shot 2016-08-30 at 12.48.27 PM.png]]

	~~/Users/reddj/Desktop/Screen Shot 2016-08-30 at 12.48.27 PM.png~~

	[[Category: Genotyping]]		[[Category: Genotyping]]

Reddj at 18:25, 30 August 2016

2016-08-30T18:25:53Z

← Older revision		Revision as of 18:25, 30 August 2016
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	[[File:Screen Shot 2016-08-30 at 12.48.27 PM.png]]		[[File:Screen Shot 2016-08-30 at 12.48.27 PM.png]]

			/Users/reddj/Desktop/Screen Shot 2016-08-30 at 12.48.27 PM.png

	[[Category: Genotyping]]		[[Category: Genotyping]]

Reddj at 18:09, 30 August 2016

2016-08-30T18:09:37Z

← Older revision		Revision as of 18:09, 30 August 2016
Line 29:		Line 29:
	For m2: the mutant band is at 150 (heterozygotes should have bands at 302, 218, and 150)		For m2: the mutant band is at 150 (heterozygotes should have bands at 302, 218, and 150)

	[[File:Screen Shot 2016-08-30 at 12.48.27 PM]]		[[File:Screen Shot 2016-08-30 at 12.48.27 PM.png]]

Reddj at 18:01, 30 August 2016

2016-08-30T18:01:59Z

← Older revision		Revision as of 18:01, 30 August 2016
Line 29:		Line 29:
	For m2: the mutant band is at 150 (heterozygotes should have bands at 302, 218, and 150)		For m2: the mutant band is at 150 (heterozygotes should have bands at 302, 218, and 150)

	Screen Shot 2016-08-30 at 12.48.27 PM		[[File:Screen Shot 2016-08-30 at 12.48.27 PM]]

Reddj at 16:50, 30 August 2016

2016-08-30T16:50:04Z

← Older revision		Revision as of 16:50, 30 August 2016
Line 28:		Line 28:
	For m1: the mutant band is at 277 (heterozygotes should have bands at 302, 218, and 277)		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)		For m2: the mutant band is at 150 (heterozygotes should have bands at 302, 218, and 150)

			Screen Shot 2016-08-30 at 12.48.27 PM



	[[Category: Genotyping]]		[[Category: Genotyping]]

Reddj: 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..."

2016-08-30T16:32:08Z

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..."

New page

==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]]