McClean: Colony PCR (Yeast) - Revision history

Megan N McClean: /* Notes */

Megan N McClean — Mon, 25 Mar 2013 19:38:20 GMT

Notes

←Older revision		Revision as of 19:38, 25 March 2013
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	'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.		'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.

-	'''*[[User:Megan N McClean\|Megan N McClean]]''' 15:36, 25 March 2013 (EDT) See [http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product] for how I prep my colony PCR ~~produts~~ for sequencing	+	'''*[[User:Megan N McClean\|Megan N McClean]]''' 15:36, 25 March 2013 (EDT) See [http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product] for how I prep my colony PCR products for sequencing

	==References==		==References==

Megan N McClean: /* Notes */

Megan N McClean — Mon, 25 Mar 2013 19:37:53 GMT

Notes

←Older revision		Revision as of 19:37, 25 March 2013
Line 74:		Line 74:
	'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.		'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.

-	'''*[[User:Megan N McClean\|Megan N McClean]]''' 15:36, 25 March 2013 (EDT) See [http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product ] for how I prep my colony PCR produts for sequencing	+	'''*[[User:Megan N McClean\|Megan N McClean]]''' 15:36, 25 March 2013 (EDT) See [http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product] for how I prep my colony PCR produts for sequencing

	==References==		==References==

Megan N McClean: /* Notes */

Megan N McClean — Mon, 25 Mar 2013 19:37:39 GMT

Notes

←Older revision		Revision as of 19:37, 25 March 2013
Line 74:		Line 74:
	'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.		'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.

-	'''*[[User:Megan N McClean\|Megan N McClean]] 15:36, 25 March 2013 (EDT) See [[http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product ]] for how I prep my colony PCR produts for sequencing	+	'''*[[User:Megan N McClean\|Megan N McClean]]''' 15:36, 25 March 2013 (EDT) See [http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product ] for how I prep my colony PCR produts for sequencing

	==References==		==References==

Megan N McClean: /* Notes */

Megan N McClean — Mon, 25 Mar 2013 19:37:17 GMT

Notes

←Older revision		Revision as of 19:37, 25 March 2013
Line 74:		Line 74:
	'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.		'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.

-	*[[User:Megan N McClean\|Megan N McClean]] 15:36, 25 March 2013 (EDT) See [[~~Sequencing Colony PCR Product \|~~ http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product]] for how I prep my colony PCR produts for sequencing	+	'''*[[User:Megan N McClean\|Megan N McClean]] 15:36, 25 March 2013 (EDT) See [[http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product ]] for how I prep my colony PCR produts for sequencing

	==References==		==References==

Megan N McClean at 19:36, 25 March 2013

Megan N McClean — Mon, 25 Mar 2013 19:36:42 GMT

←Older revision		Revision as of 19:36, 25 March 2013
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	'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.		'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.
		+
		+	*[[User:Megan N McClean\|Megan N McClean]] 15:36, 25 March 2013 (EDT) See [[Sequencing Colony PCR Product \| http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product]] for how I prep my colony PCR produts for sequencing

	==References==		==References==

Megan N McClean: /* Contact */

Megan N McClean — Tue, 04 Oct 2011 16:10:31 GMT

Contact

←Older revision		Revision as of 16:10, 4 October 2011
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	<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->		<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->

-	~~<!-- Move the relevant categories above this line to tag your protocol with the label~~	+
	[[Category:Protocol]]		[[Category:Protocol]]
		+	[[Category:Yeast]]
		+
		+	<!-- Move the relevant categories above this line to tag your protocol with the label

	[[Category:Media]]		[[Category:Media]]

-	~~[[Category:Yeast]]~~
	-->		-->

Megan N McClean: /* Notes */

Megan N McClean — Tue, 04 Oct 2011 16:09:47 GMT

Notes

←Older revision		Revision as of 16:09, 4 October 2011
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	Please feel free to post comments, questions, or improvements to this protocol.		Please feel free to post comments, questions, or improvements to this protocol.
	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		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]]''' The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly. The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone's time to figure out a shorter program. Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.		'''*[[User:Megan N McClean\|Megan N McClean]]''' The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly. The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone's time to figure out a shorter program. Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.

Megan N McClean: /* Notes */

Megan N McClean — Tue, 04 Oct 2011 16:09:26 GMT

Notes

←Older revision		Revision as of 16:09, 4 October 2011
Line 69:		Line 69:
	Please feel free to post comments, questions, or improvements to this protocol.		Please feel free to post comments, questions, or improvements to this protocol.
	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		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]]''' The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly. The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone's time to figure out a shorter program. Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.		'''*[[User:Megan N McClean\|Megan N McClean]]''' The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly. The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone's time to figure out a shorter program. Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.

Megan N McClean: /* Protocol */

Megan N McClean — Tue, 04 Oct 2011 16:08:19 GMT

Protocol

←Older revision		Revision as of 16:08, 4 October 2011
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	*4°C Forever		*4°C Forever
	**Please don't leave the thermocycler running at 4°C for longer than an hour or so, it wears out the machine. If you need to leave your PCR for longer, please change the last step of the program so that instead of holding at 4°C the program just ends (letting the samples come to room temperature). Letting the sample come to room temperature, even overnight, does not seem to cause any problems for the DNA.		**Please don't leave the thermocycler running at 4°C for longer than an hour or so, it wears out the machine. If you need to leave your PCR for longer, please change the last step of the program so that instead of holding at 4°C the program just ends (letting the samples come to room temperature). Letting the sample come to room temperature, even overnight, does not seem to cause any problems for the DNA.
		+
		+
		+
		+
		+	==='Freezing Down Positive Transformants'===
		+	Once you have run the colony PCR and confirmed which colonies are correct and which are not, take the 100μL of YPD that you set aside before, inoculate it into 4mls of fresh YPD in a test tube, and grow it to saturation overnight. Use this to freeze down glycerol stocks of the strain the following morning.

	==Notes==		==Notes==

Megan N McClean: /* Notes */

Megan N McClean — Tue, 04 Oct 2011 16:04:03 GMT

Notes

←Older revision		Revision as of 16:04, 4 October 2011
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	==Notes==		==Notes==
-	Please feel free to post comments, questions, or improvements to this protocol. ~~Happy to have your input!~~	+	Please feel free to post comments, questions, or improvements to this protocol.
-	~~#List troubleshooting tips here.~~	+
-	~~#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.~~	+
-	~~#Anecdotal observations that might be of use to others can also be posted here.~~	+
-		+
	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		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]]''' The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly. The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone's time to figure out a shorter program. Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.
		+
		+	'''*[[User:Megan N McClean\|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.

	==References==		==References==