TAS2R38 PCR amplification - Revision history

Macampbell at 12:03, 3 October 2013

2013-10-03T12:03:41Z

Macampbell at 20:42, 13 August 2012

2012-08-13T20:42:51Z

Macampbell at 20:41, 13 August 2012

2012-08-13T20:41:02Z

Macampbell at 20:30, 13 August 2012

2012-08-13T20:30:49Z

Macampbell at 20:29, 13 August 2012

2012-08-13T20:29:52Z

Macampbell at 20:26, 13 August 2012

2012-08-13T20:26:37Z

Macampbell at 14:23, 8 August 2012

2012-08-08T14:23:54Z

Macampbell at 14:55, 7 August 2012

2012-08-07T14:55:32Z

Macampbell at 14:19, 7 August 2012

2012-08-07T14:19:22Z

Macampbell at 14:18, 7 August 2012

2012-08-07T14:18:59Z

@@ Line 5: / Line 5: @@
 ! Reagent   !! Volume
 |-
-| '''extracted DNA''' || 10 µL
+| '''extracted gDNA''' || 10 µL
 |-
-| '''dH<sub>2</sub>O''' || 13 µL
+| '''Green GoTaq Mix + primers added''' || 20 µL
 |-
-| '''2X Green GoTaq Mix''' || 25 µL
+| '''Final Volume''' || 30.0 µL
 |}
 <br>

@@ Line 36: / Line 36: @@
 <center>'''References:'''<br></center>
-# [http://www.ncbi.nlm.nih.gov/gene/5726 NCBI Gene entry]
+* [http://www.ncbi.nlm.nih.gov/gene/5726 NCBI Gene entry]
-# [http://www.ncbi.nlm.nih.gov/nuccore/NG_016141.1?from=5001&to=6143&report=fasta FASTA sequence]
+* [http://www.ncbi.nlm.nih.gov/nuccore/NG_016141.1?from=5001&to=6143&report=fasta FASTA sequence]
-# [http://omim.org/entry/607751 OMIM entry (brief review and some gene structure, including SNP markers)]
+* [http://omim.org/entry/607751 OMIM entry (brief review and some gene structure, including SNP markers)]
-# [http://www.ncbi.nlm.nih.gov/pubmed/12595690 Original cloning and identification of SNP markers]
+* [http://www.ncbi.nlm.nih.gov/pubmed/12595690 Original cloning and identification of SNP markers]
-# [http://www.ncbi.nlm.nih.gov/pubmed/12584440 First identification of the TAS2R family]
+* [http://www.ncbi.nlm.nih.gov/pubmed/12584440 First identification of the TAS2R family]
-# [http://www.ncbi.nlm.nih.gov/pubmed/21036327 Overview of genetics of the chemical senses]
+* [http://www.ncbi.nlm.nih.gov/pubmed/21036327 Overview of genetics of the chemical senses]
-# [http://www.ncbi.nlm.nih.gov/pubmed/17108952 Molecular cell biology of taste]
+* [http://www.ncbi.nlm.nih.gov/pubmed/17108952 Molecular cell biology of taste]
-# [http://www.ncbi.nlm.nih.gov/pubmed/22792271 Recent cell biology]
+* [http://www.ncbi.nlm.nih.gov/pubmed/22792271 Recent cell biology]
-# [http://www.ncbi.nlm.nih.gov/pubmed/18524836 Association with nicotine dependence and alcohol consumption in African Americans 1]
+* [http://www.ncbi.nlm.nih.gov/pubmed/18524836 Association with nicotine dependence and alcohol consumption in African Americans 1]
-# [http://www.ncbi.nlm.nih.gov/pubmed/17250611 Association with nicotine dependence and alcohol consumption in African Americans 2]
+* [http://www.ncbi.nlm.nih.gov/pubmed/17250611 Association with nicotine dependence and alcohol consumption in African Americans 2]
-'''Excellent review:'''
+'''Excellent review:'''<br>
-Tepper Beverly J. 2008. Nutritional implications of genetic taste variation. Annual Review of Nutrition. 28: 367-88.
+Tepper Beverly J. 2008. Nutritional implications of genetic taste variation. Annual Review of Nutrition. 28: 367-88.<br>
+<br>
-'''Evolution and diversity:'''
+'''Evolution and diversity:'''<br>
-Kim Unkyung, Wooding Stephen, Ricci Dante, Jorde Lynn B, and Drayna Dennis. 2005. Worldwide haplotype diversity and coding sequence variation at human bitter taste receptor loci. Human Mutation. 26: 199-204.
+Kim Unkyung, Wooding Stephen, Ricci Dante, Jorde Lynn B, and Drayna Dennis. 2005. Worldwide haplotype diversity and coding sequence variation at human bitter taste receptor loci. Human Mutation. 26: 199-204.<br>
+<br>
-'''More on nutrition and food selection:'''
+'''More on nutrition and food selection:'''<br>
 * Duffy, V. B., Hayes, J. E., Davidson, A. C., Kidd, J. R., Kidd, K. K., & Bartoshuk, L. M. 2010. Vegetable intake in college-aged adults is explained by oral sensory phenotypes and TAS2R38 genotype. Chemosensory Perception, 3(3-4), 137-148.
 * Garcia-Bailo, B., Toguri, C., Eny, K. M., & El-Sohemy, A. 2009. Genetic variation in taste and its influence on food selection. Omics-a Journal of Integrative Biology, 13(1): 69-80.
 * Lipchock, S. V., Reed, D. R., & Mennella, J. A. 2012. Relationship between bitter-taste receptor genotype and solid medication formulation usage among young children: A retrospective analysis. Clinical Therapeutics, 34(3), 728-733.
 * Mennella, J., Pepino, Y., & Reed, D. 2005. Genetic and environmental determinants of bitter perception and sweet preferences. Pediatrics, 115(2), E216-E222.
+<br>
-'''Diseases:'''
+'''Diseases:'''<br>
 * Carrai, M., Steinke, V., Vodicka, P., Pardini, B., Rahner, N., Holinski-Feder, E., . . . Campa, D. 2011. Association between TAS2R38 gene polymorphisms and colorectal cancer risk: A case-control study in two independent populations of caucasian origin. Plos One, 6(6), e20464.
 * Basson MD,Bartoshuk LM,Dichello SZ,Panzini L,Weiffenbach JM,et al. 2005. Association between 6-n-propylthiouracil (PROP) bitterness and colonic neoplasms. Dig. Dis. Sci. 50:483–89.

← Older revision		Revision as of 20:41, 13 August 2012
Line 33:		Line 33:
	'''4.''' Before you can send your DNA off for sequencing, you will need to [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html clean up your PCR products]		'''4.''' Before you can send your DNA off for sequencing, you will need to [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html clean up your PCR products]

		+	'''This protocol was developed by Dr. Mark Barsoum and the 2012 RISE students.'''

−	'''~~Reference~~:'''<br>	+	<center>'''References:'''<br></center>
		+	# [http://www.ncbi.nlm.nih.gov/gene/5726 NCBI Gene entry]
		+	# [http://www.ncbi.nlm.nih.gov/nuccore/NG_016141.1?from=5001&to=6143&report=fasta FASTA sequence]
		+	# [http://omim.org/entry/607751 OMIM entry (brief review and some gene structure, including SNP markers)]
		+	# [http://www.ncbi.nlm.nih.gov/pubmed/12595690 Original cloning and identification of SNP markers]
		+	# [http://www.ncbi.nlm.nih.gov/pubmed/12584440 First identification of the TAS2R family]
		+	# [http://www.ncbi.nlm.nih.gov/pubmed/21036327 Overview of genetics of the chemical senses]
		+	# [http://www.ncbi.nlm.nih.gov/pubmed/17108952 Molecular cell biology of taste]
		+	# [http://www.ncbi.nlm.nih.gov/pubmed/22792271 Recent cell biology]
		+	# [http://www.ncbi.nlm.nih.gov/pubmed/18524836 Association with nicotine dependence and alcohol consumption in African Americans 1]
		+	# [http://www.ncbi.nlm.nih.gov/pubmed/17250611 Association with nicotine dependence and alcohol consumption in African Americans 2]
		+
		+
		+	'''Excellent review:'''
		+	Tepper Beverly J. 2008. Nutritional implications of genetic taste variation. Annual Review of Nutrition. 28: 367-88.
		+
		+	'''Evolution and diversity:'''
		+	Kim Unkyung, Wooding Stephen, Ricci Dante, Jorde Lynn B, and Drayna Dennis. 2005. Worldwide haplotype diversity and coding sequence variation at human bitter taste receptor loci. Human Mutation. 26: 199-204.
		+
		+	'''More on nutrition and food selection:'''
		+	* Duffy, V. B., Hayes, J. E., Davidson, A. C., Kidd, J. R., Kidd, K. K., & Bartoshuk, L. M. 2010. Vegetable intake in college-aged adults is explained by oral sensory phenotypes and TAS2R38 genotype. Chemosensory Perception, 3(3-4), 137-148.
		+	* Garcia-Bailo, B., Toguri, C., Eny, K. M., & El-Sohemy, A. 2009. Genetic variation in taste and its influence on food selection. Omics-a Journal of Integrative Biology, 13(1): 69-80.
		+	* Lipchock, S. V., Reed, D. R., & Mennella, J. A. 2012. Relationship between bitter-taste receptor genotype and solid medication formulation usage among young children: A retrospective analysis. Clinical Therapeutics, 34(3), 728-733.
		+	* Mennella, J., Pepino, Y., & Reed, D. 2005. Genetic and environmental determinants of bitter perception and sweet preferences. Pediatrics, 115(2), E216-E222.
		+
		+	'''Diseases:'''
		+	* Carrai, M., Steinke, V., Vodicka, P., Pardini, B., Rahner, N., Holinski-Feder, E., . . . Campa, D. 2011. Association between TAS2R38 gene polymorphisms and colorectal cancer risk: A case-control study in two independent populations of caucasian origin. Plos One, 6(6), e20464.
		+	* Basson MD,Bartoshuk LM,Dichello SZ,Panzini L,Weiffenbach JM,et al. 2005. Association between 6-n-propylthiouracil (PROP) bitterness and colonic neoplasms. Dig. Dis. Sci. 50:483–89.

@@ Line 22: / Line 22: @@
 '''PCR conditions:'''
 # 5 minutes at 95˚C.
-# 1 minute at 95˚C.
+# 30 seconds at 95˚C.
-# 1 minute at 65˚C.
+# 30 seconds at 55˚C.
-# 1 minute at 72˚C.
+# 30 seconds at 72˚C.
 # repeat steps 2 - 5 29 more times.
 # hold at room temperature.

@@ Line 5: / Line 5: @@
 ! Reagent   !! Volume
 |-
-| '''extracted DNA''' || 10.5 µL
+| '''extracted DNA''' || 10 µL
 |-
-| '''2X Green GoTaq Mix''' || 12.5 µL
+| '''dH<sub>2</sub>O''' || 13 µL
 |-
-| '''Forward Primer''' ||  1.0 µL
+| '''2X Green GoTaq Mix''' || 25 µL
 |-
-| '''Reverse Primer'''||  1.0 µL
+| '''Forward Primer''' ||  1.0 µL of 10 µM stock
 |-
-| '''Final Volume''' || 25.0 µL
+| '''Reverse Primer'''||  1.0 µL of 10 µM stock
 |}
 <br>

@@ Line 27: / Line 27: @@
 # END
-'''3.''' When the PCR is completed, store DNA frozen until you are ready to clean up the DNA in order to have your two alleles sequenced.
+'''3.''' When the PCR is completed (~2 hours), store DNA frozen until you are ready to clean up the DNA in order to have your two alleles sequenced.
 '''4.''' Before you can send your DNA off for sequencing, you will need to [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html clean up your PCR products]

← Older revision		Revision as of 14:55, 7 August 2012
Line 28:		Line 28:

	'''3.''' When the PCR is completed, store DNA frozen until you are ready to clean up the DNA in order to have your two alleles sequenced.		'''3.''' When the PCR is completed, store DNA frozen until you are ready to clean up the DNA in order to have your two alleles sequenced.
		+
		+	'''4.''' Before you can send your DNA off for sequencing, you will need to [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html clean up your PCR products]


	'''Reference:'''<br>		'''Reference:'''<br>

← Older revision		Revision as of 14:19, 7 August 2012
Line 1:		Line 1:
−	# When the DNA extraction cools ([[Isolate_genomic_DNA_from_a_single_hair_follicle]]), vortex the tubes for 30 seconds and then set up a new 500 µL microfuge tube by adding the following: <br><br>	+	'''1.''' When the DNA extraction cools ([[Isolate_genomic_DNA_from_a_single_hair_follicle]]), vortex the tubes for 30 seconds and then set up a new 500 µL microfuge tube by adding the following: <br><br>

	{\| border="1" class="wikitable"		{\| border="1" class="wikitable"

← Older revision		Revision as of 14:18, 7 August 2012
Line 28:		Line 28:

	'''3.''' When the PCR is completed, store DNA frozen until you are ready to clean up the DNA in order to have your two alleles sequenced.		'''3.''' When the PCR is completed, store DNA frozen until you are ready to clean up the DNA in order to have your two alleles sequenced.
		+
		+
		+	'''Reference:'''<br>