SHROB Genotyping - Revision history

Reddj: added SOPs

2020-06-08T17:09:21Z

added SOPs

← Older revision		Revision as of 17:09, 8 June 2020
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	__NOTOC__		__NOTOC__
			==SOP==
			*[[Safety and Animal Training]]

	==New Method==		==New Method==

Davebrid: Made older method collapsable

2016-08-09T14:01:02Z

Made older method collapsable

← Older revision		Revision as of 14:01, 9 August 2016
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			__NOTOC__

	==New Method==		==New Method==
	This method was a tetra-pair ARMS system designed using the tool at http://primer1.soton.ac.uk/primer1.html		This method was a tetra-pair ARMS system designed using the tool at http://primer1.soton.ac.uk/primer1.html

			===Primers===
	* SHROB Flanking 5': GGTTGTTTCTCAATGCAGATAGTAAATT		* SHROB Flanking 5': GGTTGTTTCTCAATGCAGATAGTAAATT
	* SHROB Inner Fwd: CCTGGACACTGTCACCTAATGATTAAA		* SHROB Inner Fwd: CCTGGACACTGTCACCTAATGATTAAA
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	* SHROB Flanking Rev: TAATACTTGTTAACATTCGAAGGGATTC		* SHROB Flanking Rev: TAATACTTGTTAACATTCGAAGGGATTC

			===Expected Results===
	There are two outer control primers and two inner primers, one of which (the Fwd primer) is specific for the mutation as shown below.		There are two outer control primers and two inner primers, one of which (the Fwd primer) is specific for the mutation as shown below.

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	\|}		\|}

			<div class="toccolours mw-collapsible mw-collapsed">
	==Original Method==		==Original Method==
	This is the older method, suggested by Takaya ''et al.''		This is the older method, suggested by Takaya ''et al.''
			<div class="mw-collapsible-content">
	===Primers===		===Primers===
	* SHROB-Fwd AGT GAA TGC TGT GCA GTC		* SHROB-Fwd AGT GAA TGC TGT GCA GTC
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	Genotyping of lean and obese Koletsky rats by PCR-RFLP analysis. Primers (5 -AGTGAATGCTGTGCAGTC-3 ; 5'-AAGGTTCTTCCATTCAAT-3') were used to amplify PCR products from 100 ng genomic DNA in a 50 uL reaction. The reaction profiles were as follows; denaturation at 94C for 30 s, annealing at 55C for 30 s, and extension at 72C for 30 s, for 30 cycles, All PCR products were digested with Tru9I, analysed by electrophoresis on a 5.0 % agarose gel (NuSieve 3:1 Agarose, Takara Shuzo Co., Ltd., Shiga, Japan), and stained with ethldium bromide. No Tru9l sites were found in the 121-bp PCR product amplified from genomic DNA derived from a wild-type lean Koletsky rat, and the PCR product was not cleaved with Tru9l (lane 1). A single Tru9l restriction site was present in the mutant allele, and Tru9I digestion of the genomic PCR products from obese Koletsky (fa/fa) rats resulted in two fragments of 82 bp and 39 bp in size (lanes 6-1 0). Tru9l digestion of the PCR products from heterozygous lean Koletsky (+/fa rats gave rise to three 121-bp,82-bp, and 39-bp fragments (lanes 2-5).		Genotyping of lean and obese Koletsky rats by PCR-RFLP analysis. Primers (5 -AGTGAATGCTGTGCAGTC-3 ; 5'-AAGGTTCTTCCATTCAAT-3') were used to amplify PCR products from 100 ng genomic DNA in a 50 uL reaction. The reaction profiles were as follows; denaturation at 94C for 30 s, annealing at 55C for 30 s, and extension at 72C for 30 s, for 30 cycles, All PCR products were digested with Tru9I, analysed by electrophoresis on a 5.0 % agarose gel (NuSieve 3:1 Agarose, Takara Shuzo Co., Ltd., Shiga, Japan), and stained with ethldium bromide. No Tru9l sites were found in the 121-bp PCR product amplified from genomic DNA derived from a wild-type lean Koletsky rat, and the PCR product was not cleaved with Tru9l (lane 1). A single Tru9l restriction site was present in the mutant allele, and Tru9I digestion of the genomic PCR products from obese Koletsky (fa/fa) rats resulted in two fragments of 82 bp and 39 bp in size (lanes 6-1 0). Tru9l digestion of the PCR products from heterozygous lean Koletsky (+/fa rats gave rise to three 121-bp,82-bp, and 39-bp fragments (lanes 2-5).
	</blockquote>		</blockquote>
			</div>
			</div>

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

Davebrid: Added schematic

2016-08-09T13:57:53Z

Added schematic

← Older revision		Revision as of 13:57, 9 August 2016
Line 9:		Line 9:
	There are two outer control primers and two inner primers, one of which (the Fwd primer) is specific for the mutation as shown below.		There are two outer control primers and two inner primers, one of which (the Fwd primer) is specific for the mutation as shown below.

			[[File:SHROB Genotyping.png]]

	There should always be a 248bp fragment, and one or both of the other fragments:		There should always be a 248bp fragment, and one or both of the other fragments:

Davebrid: Added new SHROB Genotyping protocol

2016-08-09T13:49:29Z

Added new SHROB Genotyping protocol

@@ Line 1: / Line 1: @@
-==Primers==
+==New Method==
 * SHROB-Fwd AGT GAA TGC TGT GCA GTC
 * SHROB-Rev AAG GTT CTT CCA TTC AAT
-==Reference==
+===Reference===
 From Takaya K, Ogawa Y, Hiraoka J, Hosoda K, Yamori Y, Nakao K, Koletsky RJ. Nonsense mutation of leptin receptor in the obese spontaneously hypertensive Koletsky rat. Nat Genet 14: 130–1, 1996. [10.1038/ng1096-130 doi]

Davebrid: wrote initial page

2015-12-29T20:57:51Z

wrote initial page

New page

==Primers==
* SHROB-Fwd AGT GAA TGC TGT GCA GTC
* SHROB-Rev AAG GTT CTT CCA TTC AAT

==Reference==
From Takaya K, Ogawa Y, Hiraoka J, Hosoda K, Yamori Y, Nakao K, Koletsky RJ. Nonsense mutation of leptin receptor in the obese spontaneously hypertensive Koletsky rat. Nat Genet 14: 130–1, 1996. [10.1038/ng1096-130 doi]

<blockquote>
Genotyping of lean and obese Koletsky rats by PCR-RFLP analysis. Primers (5 -AGTGAATGCTGTGCAGTC-3 ; 5'-AAGGTTCTTCCATTCAAT-3') were used to amplify PCR products from 100 ng genomic DNA in a 50 uL reaction. The reaction profiles were as follows; denaturation at 94C for 30 s, annealing at 55C for 30 s, and extension at 72C for 30 s, for 30 cycles, All PCR products were digested with Tru9I, analysed by electrophoresis on a 5.0 % agarose gel (NuSieve 3:1 Agarose, Takara Shuzo Co., Ltd., Shiga, Japan), and stained with ethldium bromide. No Tru9l sites were found in the 121-bp PCR product amplified from genomic DNA derived from a wild-type lean Koletsky rat, and the PCR product was not cleaved with Tru9l (lane 1). A single Tru9l restriction site was present in the mutant allele, and Tru9I digestion of the genomic PCR products from obese Koletsky (fa/fa) rats resulted in two fragments of 82 bp and 39 bp in size (lanes 6-1 0). Tru9l digestion of the PCR products from heterozygous lean Koletsky (+/fa rats gave rise to three 121-bp,82-bp, and 39-bp fragments (lanes 2-5).
</blockquote>

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