Ob/ob Genotyping - Revision history

Davebrid: added nots on how to make primer mixes and added band sizes

2013-09-26T19:59:19Z

added nots on how to make primer mixes and added band sizes

← Older revision		Revision as of 19:59, 26 September 2013
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	* ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3'		* ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3'
	* ob/ob - Lepob-R: 5'- GCA GAT GGA GGA GGT CTC A -3'		* ob/ob - Lepob-R: 5'- GCA GAT GGA GGA GGT CTC A -3'

			===Primer Mixes===
			Primers are resuspended at 100 uM, so add 3x10 uL of the primers to 970uL of water to make the primer mixes:
			*For '''WT Primers''' combine both RFLP primers with the WT-Lep-F primer
			*For '''Ob Primers''' combine both RFLP primers with the Lebob-R primer

	==PCR Reaction==		==PCR Reaction==
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	==Bands==		==Bands==
	~~[[File:ob_ob.jpg]]~~		There should be a control band in all lanes at 191bp
			For the wt primers if there is one or more wt alleles there should be a band at 104bp
			For the ob primers, if there is one or more ob alleles there should be a band at 123bp


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

Davebrid: changed program name

2012-10-10T15:09:00Z

changed program name

← Older revision		Revision as of 15:09, 10 October 2012
Line 8:		Line 8:

	==PCR Reaction==		==PCR Reaction==
	The PCR program is called '''ob/ob''' and is:		The PCR program is called '''ob-ob''' and is:

	* 95 °C 2 min		* 95 °C 2 min

Davebrid: added other gene specific primer

2012-10-09T16:42:52Z

added other gene specific primer

← Older revision		Revision as of 16:42, 9 October 2012
Line 5:		Line 5:
	* ob/ob RFLP-R: 5'- GCC ATC CAG GCT CTC TGG -3'		* ob/ob RFLP-R: 5'- GCC ATC CAG GCT CTC TGG -3'
	* ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3'		* ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3'
			* ob/ob - Lepob-R: 5'- GCA GAT GGA GGA GGT CTC A -3'

	==PCR Reaction==		==PCR Reaction==

Davebrid at 16:14, 9 October 2012

2012-10-09T16:14:38Z

← Older revision		Revision as of 16:14, 9 October 2012
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	** 72 °C 45 s		** 72 °C 45 s
	*72 °C 7 min		*72 °C 7 min

			==Bands==
			[[File:ob_ob.jpg]]

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

Davebrid: added categories

2012-10-09T16:03:25Z

added categories

← Older revision		Revision as of 16:03, 9 October 2012
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	** 58 °C 30 s		** 58 °C 30 s
	** 72 °C 45 s		** 72 °C 45 s
	*72 °C 7 min.		*72 °C 7 min

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

Davebrid: wrote genotyping protocol from Ellet et al

2012-10-09T16:02:55Z

wrote genotyping protocol from Ellet et al

New page

Used method described in Ellet et al (http://dx.doi.org/10.1038/oby.2008.443)

==Primers==
* ob/ob RFLP-F: 5'- TGA GTT TGT CCA AGA TGG ACC -3'
* ob/ob RFLP-R: 5'- GCC ATC CAG GCT CTC TGG -3'
* ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3'

==PCR Reaction==
The PCR program is called '''ob/ob''' and is:

* 95 °C 2 min
* 40 cycles of:
** 95 °C 30 s
** 58 °C 30 s
** 72 °C 45 s
*72 °C 7 min.