McClean: GEV Strain Construction - Revision history

Megan N McClean: /* Materials */

2012-07-02T22:27:57Z

Materials

←Older revision		Revision as of 22:27, 2 July 2012
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	==Materials==		==Materials==
-	* yMM1101 (MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2) or a similar strain with the GEV machinery driven by a constitutive promoter. Please note that this strain is DBY12020 from McIsaac, et al 2011.	+	* yMM1101 (MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2) or a similar strain with the GEV machinery driven by a constitutive promoter. Please note that this strain is DBY12020 from McIsaac, et al (2011). See reference below.
	* yMM1101 (MAT a gal10::KanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (BY4742)) genomic DNA (see Notes)		* yMM1101 (MAT a gal10::KanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (BY4742)) genomic DNA (see Notes)
	* Primers (see below for how to design)		* Primers (see below for how to design)

Megan N McClean: /* Materials */

2012-07-02T22:27:31Z

Materials

←Older revision		Revision as of 22:27, 2 July 2012
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	* yMM1101 (MAT a gal10::KanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (BY4742)) genomic DNA (see Notes)		* yMM1101 (MAT a gal10::KanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (BY4742)) genomic DNA (see Notes)
	* Primers (see below for how to design)		* Primers (see below for how to design)
-	* PCR supplies ()	+	* PCR supplies (see: [[McClean: Takara PrimeStar PCR \| Takara PrimeStar PCR]])
-	* Transformation supplies ()	+	* Transformation supplies (see: [[McClean:Yeast Transformation \| Yeast Transformation (S. cerevisiae)]])

	==Protocol==		==Protocol==

Megan N McClean: /* Contact */

2012-07-02T22:04:33Z

Contact

←Older revision		Revision as of 22:04, 2 July 2012
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	==Contact==		==Contact==
	<!--Change the information below to your info if you add a new protocol-->		<!--Change the information below to your info if you add a new protocol-->
-	*'''[[User:Megan N McClean\|Megan N McClean]] 14:01, 20 July ~~2011~~ (EDT)'''	+	*'''[[User:Megan N McClean\|Megan N McClean]] 14:01, 2 July 2012 (EDT)'''

	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

Megan N McClean: /* Notes */

2012-07-02T22:04:07Z

Notes

←Older revision		Revision as of 22:04, 2 July 2012
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	<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->		<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-	*[[User:Megan N McClean\|Megan N McClean]] 17:48, 29 June 2012 (EDT) I am incredibly lazy and use the [[McClean: Genomic DNA Prep (Bust 'n' Grab Protocol)\| Bust n' Grab]] ~~"Bust n' Grab"~~ protocol for genomic DNA prep. To be even lazier, I never measure the concentration of the genomic DNA that comes out of the protocol, I just dilute it 1:100 with water and use 1μL of that in a 50μL PCR reaction (using Takara polymerase). This almost always works. When it doesn't work it is usually the concentration of the genomic DNA that is the problem, so I just go back and try 1:10 and 1:1000 dilutions. Feel free to not be this lazy, but this is how I do it and it usually works.	+	*[[User:Megan N McClean\|Megan N McClean]] 17:48, 29 June 2012 (EDT) I am incredibly lazy and use the [[McClean: Genomic DNA Prep (Bust 'n' Grab Protocol)\| Bust n' Grab]] protocol for genomic DNA prep. To be even lazier, I never measure the concentration of the genomic DNA that comes out of the protocol, I just dilute it 1:100 with water and use 1μL of that in a 50μL PCR reaction (using Takara PrimeStar polymerase). This almost always works. When it doesn't work it is usually the concentration of the genomic DNA that is the problem, so I just go back and try 1:10 and 1:1000 dilutions. Feel free to not be this lazy, but this is how I do it and it usually works.

	==References==		==References==

Megan N McClean: /* Notes */

2012-07-02T22:03:25Z

Notes

←Older revision		Revision as of 22:03, 2 July 2012
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	<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->		<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-	*[[User:Megan N McClean\|Megan N McClean]] 17:48, 29 June 2012 (EDT) I am incredibly lazy and use the [[McClean: Genomic DNA Prep (Bust 'n' Grab Protocol) Bust n' Grab]] "Bust n' Grab" protocol for genomic DNA prep. To be even lazier, I never measure the concentration of the genomic DNA that comes out of the protocol, I just dilute it 1:100 with water and use 1μL of that in a 50μL PCR reaction (using Takara polymerase). This almost always works. When it doesn't work it is usually the concentration of the genomic DNA that is the problem, so I just go back and try 1:10 and 1:1000 dilutions. Feel free to not be this lazy, but this is how I do it and it usually works.	+	*[[User:Megan N McClean\|Megan N McClean]] 17:48, 29 June 2012 (EDT) I am incredibly lazy and use the [[McClean: Genomic DNA Prep (Bust 'n' Grab Protocol)\| Bust n' Grab]] "Bust n' Grab" protocol for genomic DNA prep. To be even lazier, I never measure the concentration of the genomic DNA that comes out of the protocol, I just dilute it 1:100 with water and use 1μL of that in a 50μL PCR reaction (using Takara polymerase). This almost always works. When it doesn't work it is usually the concentration of the genomic DNA that is the problem, so I just go back and try 1:10 and 1:1000 dilutions. Feel free to not be this lazy, but this is how I do it and it usually works.

	==References==		==References==

Megan N McClean: /* Materials */

2012-06-29T23:48:07Z

Materials

←Older revision		Revision as of 23:48, 29 June 2012
Line 11:		Line 11:
	* yMM1101 (MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2) or a similar strain with the GEV machinery driven by a constitutive promoter. Please note that this strain is DBY12020 from McIsaac, et al 2011.		* yMM1101 (MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2) or a similar strain with the GEV machinery driven by a constitutive promoter. Please note that this strain is DBY12020 from McIsaac, et al 2011.
	* yMM1101 (MAT a gal10::KanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (BY4742)) genomic DNA (see Notes)		* yMM1101 (MAT a gal10::KanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (BY4742)) genomic DNA (see Notes)
		+	* Primers (see below for how to design)
		+	* PCR supplies ()
		+	* Transformation supplies ()

	==Protocol==		==Protocol==

Megan N McClean: /* Overview */

2012-06-29T23:47:22Z

Overview

←Older revision		Revision as of 23:47, 29 June 2012
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	==Overview==		==Overview==
	This protocol explains how to insert the GAL1 promoter in front of a gene of interest in the appropriate strain with the GEV machinery (yMM1101 MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2 or similar) so that production of the transcript (and therefore the protein) is inducible with β-estradiol.		This protocol explains how to insert the GAL1 promoter in front of a gene of interest in the appropriate strain with the GEV machinery (yMM1101 MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2 or similar) so that production of the transcript (and therefore the protein) is inducible with β-estradiol.
		+
		+	Primarily this protocol deals with how to appropriately design primers to insert the GAL1 promoter. Other parts of the protocol (PCR, transformation) are standard and you are referred to other McClean lab protocols for those bits.

	This protocol has been modified from Scott McIsaac's (Botstein Lab) protocol. Use of this protocol and the subsequent strains should cite his paper (see references below).		This protocol has been modified from Scott McIsaac's (Botstein Lab) protocol. Use of this protocol and the subsequent strains should cite his paper (see references below).

Megan N McClean: /* Overview */

2012-06-29T22:52:08Z

Overview

←Older revision		Revision as of 22:52, 29 June 2012
Line 2:		Line 2:

	==Overview==		==Overview==
-	This protocol explains how to insert the GAL1 promoter in front of a gene of interest in the appropriate strain with the GEV machinery (yMM1101 MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2 or similar) so that production of the transcript (and therefore the protein) is inducible with ~~\{beta}~~-estradiol.	+	This protocol explains how to insert the GAL1 promoter in front of a gene of interest in the appropriate strain with the GEV machinery (yMM1101 MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2 or similar) so that production of the transcript (and therefore the protein) is inducible with β-estradiol.

	This protocol has been modified from Scott McIsaac's (Botstein Lab) protocol. Use of this protocol and the subsequent strains should cite his paper (see references below).		This protocol has been modified from Scott McIsaac's (Botstein Lab) protocol. Use of this protocol and the subsequent strains should cite his paper (see references below).

Megan N McClean: /* Overview */

2012-06-29T22:51:38Z

Overview

←Older revision		Revision as of 22:51, 29 June 2012
Line 2:		Line 2:

	==Overview==		==Overview==
-	This protocol explains how to insert the GAL1 promoter in front of a gene of interest in the appropriate strain with the GEV machinery (yMM1101 MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2 or similar)	+	This protocol explains how to insert the GAL1 promoter in front of a gene of interest in the appropriate strain with the GEV machinery (yMM1101 MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2 or similar) so that production of the transcript (and therefore the protein) is inducible with \{beta}-estradiol.

	This protocol has been modified from Scott McIsaac's (Botstein Lab) protocol. Use of this protocol and the subsequent strains should cite his paper (see references below).		This protocol has been modified from Scott McIsaac's (Botstein Lab) protocol. Use of this protocol and the subsequent strains should cite his paper (see references below).

Megan N McClean: /* Notes */

2012-06-29T22:30:09Z

Notes

←Older revision		Revision as of 22:30, 29 June 2012
Line 29:		Line 29:
	<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->		<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-	*[[User:Megan N McClean\|Megan N McClean]] 17:48, 29 June 2012 (EDT) I am incredibly lazy and use the [McClean: Genomic DNA Prep (Bust 'n' Grab Protocol) Bust n' Grab] "Bust n' Grab" protocol for genomic DNA prep. To be even lazier, I never measure the concentration of the genomic DNA that comes out of the protocol, I just dilute it 1:100 with water and use 1μL of that in a 50μL PCR reaction (using Takara polymerase). This almost always works. When it doesn't work it is usually the concentration of the genomic DNA that is the problem, so I just go back and try 1:10 and 1:1000 dilutions. Feel free to not be this lazy, but this is how I do it and it usually works.	+	*[[User:Megan N McClean\|Megan N McClean]] 17:48, 29 June 2012 (EDT) I am incredibly lazy and use the [[McClean: Genomic DNA Prep (Bust 'n' Grab Protocol) Bust n' Grab]] "Bust n' Grab" protocol for genomic DNA prep. To be even lazier, I never measure the concentration of the genomic DNA that comes out of the protocol, I just dilute it 1:100 with water and use 1μL of that in a 50μL PCR reaction (using Takara polymerase). This almost always works. When it doesn't work it is usually the concentration of the genomic DNA that is the problem, so I just go back and try 1:10 and 1:1000 dilutions. Feel free to not be this lazy, but this is how I do it and it usually works.

	==References==		==References==