McClean: Yeast Mating Halo Assay - Revision history

Megan N McClean at 19:54, 15 March 2013

Megan N McClean — Fri, 15 Mar 2013 19:54:52 GMT

←Older revision		Revision as of 19:54, 15 March 2013
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	*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see image), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.		*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see image), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.
	[[Image:YeastMatingAssay.png\|thumb\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]		[[Image:YeastMatingAssay.png\|thumb\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]
-	//[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]	+	<!-- [[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]] -->
		+
	<br style="clear:both;"/>		<br style="clear:both;"/>

Megan N McClean: /* Notes */

Megan N McClean — Fri, 15 Mar 2013 19:54:06 GMT

Notes

←Older revision		Revision as of 19:54, 15 March 2013
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	*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see image), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.		*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see image), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.
-		+	[[Image:YeastMatingAssay.png\|thumb\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]
-	[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]	+	//[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]
	<br style="clear:both;"/>		<br style="clear:both;"/>

Megan N McClean: /* Notes */

Megan N McClean — Fri, 15 Mar 2013 19:49:17 GMT

Notes

←Older revision		Revision as of 19:49, 15 March 2013
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	*[[User:Megan N McClean\|Megan N McClean]] 14:37, 23 September 2012 (EDT) The yMM422 and 421 strains both showed good halos for me this time. Here is what I did (granted, this was very sloppy because I was in a rush, so I am surprised it worked as well as it did): I took several colonies of yMM422 and yMM421 off of a very old YPD plate they had been struck onto and put them into about 1ml of liquid YPD. I picked the lightest red colonies. I'm guessing just from the shade of the YPD after I had mixed the colonies into it that the OD600 was around 0.8-1. I immediately took 200μL of this and spread it using glass beads onto a YPD plate. This I allowed to dry for 30 minutes at 30°C. Once it was dry I frogged the tetrads onto it, and left it for ~24 hours growing at 30°C. One of the yMM422 plates worked well (the drier one) and the other one that didn't quite dry before I frogged onto it only shows a few clear halos...perhaps because there are some places where the yMM422 lawn is too thick?		*[[User:Megan N McClean\|Megan N McClean]] 14:37, 23 September 2012 (EDT) The yMM422 and 421 strains both showed good halos for me this time. Here is what I did (granted, this was very sloppy because I was in a rush, so I am surprised it worked as well as it did): I took several colonies of yMM422 and yMM421 off of a very old YPD plate they had been struck onto and put them into about 1ml of liquid YPD. I picked the lightest red colonies. I'm guessing just from the shade of the YPD after I had mixed the colonies into it that the OD600 was around 0.8-1. I immediately took 200μL of this and spread it using glass beads onto a YPD plate. This I allowed to dry for 30 minutes at 30°C. Once it was dry I frogged the tetrads onto it, and left it for ~24 hours growing at 30°C. One of the yMM422 plates worked well (the drier one) and the other one that didn't quite dry before I frogged onto it only shows a few clear halos...perhaps because there are some places where the yMM422 lawn is too thick?

-	*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see ~~images~~), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.	+	*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see image), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.

	[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]		[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]

Megan N McClean: /* Notes */

Megan N McClean — Fri, 15 Mar 2013 19:48:43 GMT

Notes

←Older revision		Revision as of 19:48, 15 March 2013
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	[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]		[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]
		+	<br style="clear:both;"/>

	==Contact==		==Contact==

Megan N McClean: /* Notes */

Megan N McClean — Fri, 15 Mar 2013 19:47:53 GMT

Notes

←Older revision		Revision as of 19:47, 15 March 2013
Line 38:		Line 38:

	*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see images), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.		*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see images), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.
		+
		+	[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]

	==Contact==		==Contact==

Megan N McClean: /* Contact */

Megan N McClean — Fri, 15 Mar 2013 19:44:30 GMT

Contact

←Older revision		Revision as of 19:44, 15 March 2013
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	<!--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, 03 November 11(EDT)'''		*'''[[User:Megan N McClean\|Megan N McClean]] 14:01, 03 November 11(EDT)'''
-	*'''[[User:Jue Wang\|Jue Wang]] 14:01, 15 March 13(EDT)'''	+	*'''[[User:Jue Wang\|Jue Wang]] 15:44, 15 March 13(EDT)'''
	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

Megan N McClean: /* Contact */

Megan N McClean — Fri, 15 Mar 2013 19:43:59 GMT

Contact

←Older revision		Revision as of 19:43, 15 March 2013
Line 42:		Line 42:
	<!--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, 03 November 11(EDT)'''		*'''[[User:Megan N McClean\|Megan N McClean]] 14:01, 03 November 11(EDT)'''
-		+	*'''[[User:Jue Wang\|Jue Wang]] 14:01, 15 March 13(EDT)'''
	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

Megan N McClean at 19:42, 15 March 2013

Megan N McClean — Fri, 15 Mar 2013 19:42:50 GMT

←Older revision		Revision as of 19:42, 15 March 2013
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	mating type, the cells arrest.		mating type, the cells arrest.

		+	Elia, L. and Marsh, L. (1996) Role of the ABC transporter Ste6 in cell fusion during yeast conjugation ''J Cell Biol'' '''135''' 741-751

	==Notes==		==Notes==

Megan N McClean: /* Notes */

Megan N McClean — Fri, 15 Mar 2013 19:40:56 GMT

Notes

←Older revision		Revision as of 19:40, 15 March 2013
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	*[[User:Megan N McClean\|Megan N McClean]] 11:02, 3 November 2011 (EDT) We have been having some trouble lately with the yMM422 strain. Strains of the opposite mating type have not been forming scorable halos. We may want to try fresh stock from the Botstein lab's -80°C stock. Alternatively, it seems to work 'sometimes' and since none of us are that careful about how many cells we seed or what growth phase they are in when we do so, perhaps we should play around with that and then be consistent in the future.		*[[User:Megan N McClean\|Megan N McClean]] 11:02, 3 November 2011 (EDT) We have been having some trouble lately with the yMM422 strain. Strains of the opposite mating type have not been forming scorable halos. We may want to try fresh stock from the Botstein lab's -80°C stock. Alternatively, it seems to work 'sometimes' and since none of us are that careful about how many cells we seed or what growth phase they are in when we do so, perhaps we should play around with that and then be consistent in the future.

-	*[[User:Megan N McClean\|Megan N McClean]] 14:37, 23 September 2012 (EDT) The yMM422 and 421 strains both showed good halos for me this time. Here is what I did (granted, this was very sloppy because I was in a rush, so I am surprised it worked as well as it did): I took several colonies of yMM422 and yMM421 off of a very old YPD plate they had been struck onto and put them into about 1ml of liquid YPD. I picked the lightest red colonies. I'm guessing just from the shade of the YPD after I had mixed the colonies into it that the OD600 was around 0.8-1. I immediately took 200μL of this and spread it using glass beads onto a YPD plate. This I allowed to dry for 30 minutes at 30°C. Once it was dry I frogged the tetrads onto it, and left it for ~24 hours growing at 30°C. One of the yMM422 plates worked well (the ~~dryer~~ one) and the other one that didn't quite dry before I frogged onto it only shows a few clear halos...perhaps because there are some places where the yMM422 lawn is too thick?	+	*[[User:Megan N McClean\|Megan N McClean]] 14:37, 23 September 2012 (EDT) The yMM422 and 421 strains both showed good halos for me this time. Here is what I did (granted, this was very sloppy because I was in a rush, so I am surprised it worked as well as it did): I took several colonies of yMM422 and yMM421 off of a very old YPD plate they had been struck onto and put them into about 1ml of liquid YPD. I picked the lightest red colonies. I'm guessing just from the shade of the YPD after I had mixed the colonies into it that the OD600 was around 0.8-1. I immediately took 200μL of this and spread it using glass beads onto a YPD plate. This I allowed to dry for 30 minutes at 30°C. Once it was dry I frogged the tetrads onto it, and left it for ~24 hours growing at 30°C. One of the yMM422 plates worked well (the drier one) and the other one that didn't quite dry before I frogged onto it only shows a few clear halos...perhaps because there are some places where the yMM422 lawn is too thick?
		+
		+	*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see images), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.

	==Contact==		==Contact==

Megan N McClean: /* Materials */

Megan N McClean — Fri, 15 Mar 2013 19:24:18 GMT

Materials

←Older revision		Revision as of 19:24, 15 March 2013
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	* yMM422 (aka DBY7442, XT1-20A) MATalpha leu- ura- ade- sst2		* yMM422 (aka DBY7442, XT1-20A) MATalpha leu- ura- ade- sst2
	* YPD plates		* YPD plates
-	* YPD + 0.05% Triton X-100 (see ~~Note~~ below)	+	* YPD + 0.05% Triton X-100 (see Notes below)

	==Protocol==		==Protocol==

←Older revision		Revision as of 19:49, 15 March 2013
Line 37:		Line 37:
	*[[User:Megan N McClean\|Megan N McClean]] 14:37, 23 September 2012 (EDT) The yMM422 and 421 strains both showed good halos for me this time. Here is what I did (granted, this was very sloppy because I was in a rush, so I am surprised it worked as well as it did): I took several colonies of yMM422 and yMM421 off of a very old YPD plate they had been struck onto and put them into about 1ml of liquid YPD. I picked the lightest red colonies. I'm guessing just from the shade of the YPD after I had mixed the colonies into it that the OD600 was around 0.8-1. I immediately took 200μL of this and spread it using glass beads onto a YPD plate. This I allowed to dry for 30 minutes at 30°C. Once it was dry I frogged the tetrads onto it, and left it for ~24 hours growing at 30°C. One of the yMM422 plates worked well (the drier one) and the other one that didn't quite dry before I frogged onto it only shows a few clear halos...perhaps because there are some places where the yMM422 lawn is too thick?		*[[User:Megan N McClean\|Megan N McClean]] 14:37, 23 September 2012 (EDT) The yMM422 and 421 strains both showed good halos for me this time. Here is what I did (granted, this was very sloppy because I was in a rush, so I am surprised it worked as well as it did): I took several colonies of yMM422 and yMM421 off of a very old YPD plate they had been struck onto and put them into about 1ml of liquid YPD. I picked the lightest red colonies. I'm guessing just from the shade of the YPD after I had mixed the colonies into it that the OD600 was around 0.8-1. I immediately took 200μL of this and spread it using glass beads onto a YPD plate. This I allowed to dry for 30 minutes at 30°C. Once it was dry I frogged the tetrads onto it, and left it for ~24 hours growing at 30°C. One of the yMM422 plates worked well (the drier one) and the other one that didn't quite dry before I frogged onto it only shows a few clear halos...perhaps because there are some places where the yMM422 lawn is too thick?

-	*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see ~~images~~), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.	+	*[[User:Megan N McClean\|Megan N McClean]] 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see image), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.

	[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]		[[Image:YeastMatingAssay.png\|150px\|right\|Mating Assay with yMM422, Courtesy of Jue Wang]]