Sex genotyping of mice - Revision history

Jakob Suckale: test PCR

Jakob Suckale — Thu, 28 Apr 2011 09:50:04 GMT

test PCR

←Older revision		Revision as of 09:50, 28 April 2011
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	: Y chromosomal SmcY = Kdm5d [http://www.ncbi.nlm.nih.gov/gene/20592]		: Y chromosomal SmcY = Kdm5d [http://www.ncbi.nlm.nih.gov/gene/20592]

		+
		+	== Test PCR with adult tail genomic samples ==
		+
		+	[[Image:Mouse sex genotyping.png\|500px]]
		+
		+	'''Conditions''': Templates are known adult male and female tail gDNA samples. Standard PCR cycled using progressively lowered annealing temperature (65-58°C for first cycles, then 58°C); same conditions for all reactions. Primers as listed above. 12μL of a 25μL + 5μL Orange G run on a 2% agarose gel.
		+
		+	'''Interpretation''': The ''sry F/R'' pair worked best but does show weak unspecific products of similar size as the male-specific 273bp band. The sry 1/2 pair is probably also specific but bands are harder to recognise. For the SMC, we didn't observe the described doublet expected in males.
		+
		+	Keep in mind that this is a standard PCR. Amplification and specificity can probably be increased by optimisation of the PCR reaction.

Jakob Suckale at 13:28, 6 April 2011

Jakob Suckale — Wed, 06 Apr 2011 13:28:37 GMT

←Older revision		Revision as of 13:28, 6 April 2011
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	* SMCX-1 5'-CCGCTGCCAAATTCTTTGG-3'		* SMCX-1 5'-CCGCTGCCAAATTCTTTGG-3'
	* SMC4-1 5'-TGAAGCTTTTGGCTTTGAG-3'		* SMC4-1 5'-TGAAGCTTTTGGCTTTGAG-3'
-	* PCR product: females single band, males 2 bands because of an intron difference between the X and Y genes (Agulnik 1997, PMID 9060413); ~~source:~~ [http://ko.cwru.edu/services/musfrming.html]	+	* PCR product: females single band, males 2 bands because of an intron difference between the X and Y genes (Agulnik 1997, PMID 9060413); adapted from [http://ko.cwru.edu/services/musfrming.html Case Western Transgenic Facility]

	: X chromosomal SmcX = Kdm5c [http://www.ncbi.nlm.nih.gov/gene/20591]		: X chromosomal SmcX = Kdm5c [http://www.ncbi.nlm.nih.gov/gene/20591]

Jakob Suckale at 13:26, 6 April 2011

Jakob Suckale — Wed, 06 Apr 2011 13:26:41 GMT

Jakob Suckale at 13:25, 6 April 2011

Jakob Suckale — Wed, 06 Apr 2011 13:25:22 GMT

←Older revision		Revision as of 13:25, 6 April 2011
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-	[Category:Protocol]	+	[[Category:Protocol]]
-	[Category:PCR]	+	[[Category:PCR]]
-	[Category:Mouse]	+	[[Category:Mouse]]

Jakob Suckale: categories

Jakob Suckale — Wed, 06 Apr 2011 13:25:03 GMT

Jakob Suckale: SmcX/SmcY

Jakob Suckale — Wed, 06 Apr 2011 13:24:22 GMT

SmcX/SmcY

←Older revision		Revision as of 13:24, 6 April 2011
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	* PCR product: 166, 146 bp doublet		* PCR product: 166, 146 bp doublet
	: [http://www.mmrrc.org/strains/10318/di_protocol.pdf MMRRC's genotyping protocol including Sry primers]		: [http://www.mmrrc.org/strains/10318/di_protocol.pdf MMRRC's genotyping protocol including Sry primers]
		+
		+
		+	== Homologous pair SmcX/SmcY ==
		+
		+	* SMCX-1 5'CCGCTGCCAAATTCTTTGG3'
		+	* SMC4-1 5'TGAAGCTTTTGGCTTTGAG3'
		+	* PCR product: females single band, males 2 bands because of an intron difference between the X and Y genes (Agulnik 1997, PMID 9060413); source: [http://ko.cwru.edu/services/musfrming.html]
		+
		+	: X chromosomal SmcX = Kdm5c [http://www.ncbi.nlm.nih.gov/gene/20591]
		+	: Y chromosomal SmcY = Kdm5d [http://www.ncbi.nlm.nih.gov/gene/20592]

Jakob Suckale at 13:00, 6 April 2011

Jakob Suckale — Wed, 06 Apr 2011 13:00:04 GMT

←Older revision		Revision as of 13:00, 6 April 2011
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	If the sex cannot be determined by inspection, a genotyping PCR for any region only found on the Y chromosome [http://tinyurl.com/65hwmtj] can be used.		If the sex cannot be determined by inspection, a genotyping PCR for any region only found on the Y chromosome [http://tinyurl.com/65hwmtj] can be used.

-	=== Common male-specific PCRs ===	+	== Common male-specific PCRs ==

-	===='''Sry''' Sex-determining region of the Y chromosome [http://www.ncbi.nlm.nih.gov/gene/21674] ====	+	==='''Sry''' Sex-determining region of the Y chromosome [http://www.ncbi.nlm.nih.gov/gene/21674] ===
	* forward SRY F : TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA		* forward SRY F : TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA
	* reverse SRY R : CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA		* reverse SRY R : CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA
-	: PCR product: 273 base pair PCR product	+	* PCR product: 273 base pair PCR product
-	[http://mgc.wustl.edu/Protocols/PCRGenotypingPrimerPairs/tabid/154/Default.aspx Uni Washington, St. Louis, Mouse Genetics Core]	+	: [http://mgc.wustl.edu/Protocols/PCRGenotypingPrimerPairs/tabid/154/Default.aspx Uni Washington, St. Louis, Mouse Genetics Core]

	* forward sry1 AACAACTGGGCTTTGCACATTG		* forward sry1 AACAACTGGGCTTTGCACATTG
	* reverse sry2 GTTTATCAGGGTTTCTCTCTAGC		* reverse sry2 GTTTATCAGGGTTTCTCTCTAGC
-	: PCR product: 166, 146 bp doublet	+	* PCR product: 166, 146 bp doublet
-	[http://www.mmrrc.org/strains/10318/di_protocol.pdf MMRRC's genotyping protocol including Sry primers]	+	: [http://www.mmrrc.org/strains/10318/di_protocol.pdf MMRRC's genotyping protocol including Sry primers]

Jakob Suckale: Sry PCR

Jakob Suckale — Wed, 06 Apr 2011 12:59:14 GMT

Sry PCR

←Older revision		Revision as of 12:59, 6 April 2011
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	If the sex cannot be determined by inspection, a genotyping PCR for any region only found on the Y chromosome [http://tinyurl.com/65hwmtj] can be used.		If the sex cannot be determined by inspection, a genotyping PCR for any region only found on the Y chromosome [http://tinyurl.com/65hwmtj] can be used.
		+
		+	=== Common male-specific PCRs ===
		+
		+	===='''Sry''' Sex-determining region of the Y chromosome [http://www.ncbi.nlm.nih.gov/gene/21674] ====
		+	* forward SRY F : TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA
		+	* reverse SRY R : CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA
		+	: PCR product: 273 base pair PCR product
		+	[http://mgc.wustl.edu/Protocols/PCRGenotypingPrimerPairs/tabid/154/Default.aspx Uni Washington, St. Louis, Mouse Genetics Core]
		+
		+	* forward sry1 AACAACTGGGCTTTGCACATTG
		+	* reverse sry2 GTTTATCAGGGTTTCTCTCTAGC
		+	: PCR product: 166, 146 bp doublet
		+	[http://www.mmrrc.org/strains/10318/di_protocol.pdf MMRRC's genotyping protocol including Sry primers]

Jakob Suckale at 12:51, 6 April 2011

Jakob Suckale — Wed, 06 Apr 2011 12:51:37 GMT

←Older revision		Revision as of 12:51, 6 April 2011
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	While adult mice can be readily sexed using anatomical features, young male and female mouse embryos are not yet sufficiently developed to assess their sex. In fact, their genital tubercles look superficially identical. At E16, only about 4 days before birth, the tubercle looks like this: [http://books.google.com/books?id=RlnTPjsBFuQC&lpg=PA108&dq=baskin%20male%20female%20genital%20tubercle&pg=PA108#v=onepage&q=baskin%20male%20female%20genital%20tubercle&f=false E16 genital tubercles in Baskin's book, p. 108, figure 2].		While adult mice can be readily sexed using anatomical features, young male and female mouse embryos are not yet sufficiently developed to assess their sex. In fact, their genital tubercles look superficially identical. At E16, only about 4 days before birth, the tubercle looks like this: [http://books.google.com/books?id=RlnTPjsBFuQC&lpg=PA108&dq=baskin%20male%20female%20genital%20tubercle&pg=PA108#v=onepage&q=baskin%20male%20female%20genital%20tubercle&f=false E16 genital tubercles in Baskin's book, p. 108, figure 2].
		+
		+	If the sex cannot be determined by inspection, a genotyping PCR for any region only found on the Y chromosome [http://tinyurl.com/65hwmtj] can be used.

Jakob Suckale: * page created *

Jakob Suckale — Wed, 06 Apr 2011 12:41:52 GMT

*** page created ***

New page

While adult mice can be readily sexed using anatomical features, young male and female mouse embryos are not yet sufficiently developed to assess their sex. In fact, their genital tubercles look superficially identical. At E16, only about 4 days before birth, the tubercle looks like this: [http://books.google.com/books?id=RlnTPjsBFuQC&lpg=PA108&dq=baskin%20male%20female%20genital%20tubercle&pg=PA108#v=onepage&q=baskin%20male%20female%20genital%20tubercle&f=false E16 genital tubercles in Baskin's book, p. 108, figure 2].

←Older revision		Revision as of 13:26, 6 April 2011
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	If the sex cannot be determined by inspection, a genotyping PCR for any region only found on the Y chromosome [http://tinyurl.com/65hwmtj] can be used.		If the sex cannot be determined by inspection, a genotyping PCR for any region only found on the Y chromosome [http://tinyurl.com/65hwmtj] can be used.
		+

	== Common male-specific PCRs ==		== Common male-specific PCRs ==
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	* forward SRY F : TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA		* forward SRY F : TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA
	* reverse SRY R : CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA		* reverse SRY R : CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA
-	* PCR product: 273 base pair ~~PCR product~~	+	* PCR product: 273 base pair
	: [http://mgc.wustl.edu/Protocols/PCRGenotypingPrimerPairs/tabid/154/Default.aspx Uni Washington, St. Louis, Mouse Genetics Core]		: [http://mgc.wustl.edu/Protocols/PCRGenotypingPrimerPairs/tabid/154/Default.aspx Uni Washington, St. Louis, Mouse Genetics Core]

	* forward sry1 AACAACTGGGCTTTGCACATTG		* forward sry1 AACAACTGGGCTTTGCACATTG
	* reverse sry2 GTTTATCAGGGTTTCTCTCTAGC		* reverse sry2 GTTTATCAGGGTTTCTCTCTAGC
-	* PCR product: ~~166,~~ 146 bp doublet	+	* PCR product: 146/166 bp doublet
	: [http://www.mmrrc.org/strains/10318/di_protocol.pdf MMRRC's genotyping protocol including Sry primers]		: [http://www.mmrrc.org/strains/10318/di_protocol.pdf MMRRC's genotyping protocol including Sry primers]

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	== Homologous pair SmcX/SmcY ==		== Homologous pair SmcX/SmcY ==

-	* SMCX-1 5'~~CCGCTGCCAAATTCTTTGG3~~'	+	* SMCX-1 5'-CCGCTGCCAAATTCTTTGG-3'
-	* SMC4-1 5'~~TGAAGCTTTTGGCTTTGAG3~~'	+	* SMC4-1 5'-TGAAGCTTTTGGCTTTGAG-3'
	* PCR product: females single band, males 2 bands because of an intron difference between the X and Y genes (Agulnik 1997, PMID 9060413); source: [http://ko.cwru.edu/services/musfrming.html]		* PCR product: females single band, males 2 bands because of an intron difference between the X and Y genes (Agulnik 1997, PMID 9060413); source: [http://ko.cwru.edu/services/musfrming.html]

	: X chromosomal SmcX = Kdm5c [http://www.ncbi.nlm.nih.gov/gene/20591]		: X chromosomal SmcX = Kdm5c [http://www.ncbi.nlm.nih.gov/gene/20591]
	: Y chromosomal SmcY = Kdm5d [http://www.ncbi.nlm.nih.gov/gene/20592]		: Y chromosomal SmcY = Kdm5d [http://www.ncbi.nlm.nih.gov/gene/20592]
		+

←Older revision		Revision as of 13:25, 6 April 2011
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	: X chromosomal SmcX = Kdm5c [http://www.ncbi.nlm.nih.gov/gene/20591]		: X chromosomal SmcX = Kdm5c [http://www.ncbi.nlm.nih.gov/gene/20591]
	: Y chromosomal SmcY = Kdm5d [http://www.ncbi.nlm.nih.gov/gene/20592]		: Y chromosomal SmcY = Kdm5d [http://www.ncbi.nlm.nih.gov/gene/20592]
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		+
		+	[Category:Protocol]
		+	[Category:PCR]
		+	[Category:Mouse]