Lidstrom:Overlap Extension PCR - Revision history

Janet B. Matsen: /* If you are sewing larger pieces */

2013-03-08T21:55:15Z

If you are sewing larger pieces

←Older revision		Revision as of 21:55, 8 March 2013
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	== If you are sewing larger pieces ==		== If you are sewing larger pieces ==
-	* The paper is key reading.	+	* The paper ** For longer fragments: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373371/?tool=pmcentrez&rendertype=abstract Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously] is key reading.
		+	* Key points:
		+	** use high (68oC) annealing temps
		+	** don't gel purify PCR products you plan to sew
		+	*** Their experience lead them to believe this is a deal breaker; they rationalized it by DNA damage cause by ethidium bromide and the UV exposure.
		+	** You may need nested primers to amplify the product in the + primer reaction.
		+	*** This is bad news if you are trying to sew Gibson assembly pieces together.

	== Use Primers on "Sewing PCR" product ==		== Use Primers on "Sewing PCR" product ==

Janet B. Matsen: /* Use Primers on "Sewing PCR" product */

2013-03-08T21:50:12Z

Use Primers on "Sewing PCR" product

←Older revision		Revision as of 21:50, 8 March 2013
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	* You can also do the next PCR step without examining on a gel.		* You can also do the next PCR step without examining on a gel.
	** Janet recommends proceeding to this step unless you have problems. If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.		** Janet recommends proceeding to this step unless you have problems. If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.
		+
		+	== If you are sewing larger pieces ==
		+	* The paper is key reading.

	== Use Primers on "Sewing PCR" product ==		== Use Primers on "Sewing PCR" product ==

Janet B. Matsen: /* The Big Picture */

2013-03-08T21:49:40Z

The Big Picture

←Older revision		Revision as of 21:49, 8 March 2013
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	== The Big Picture ==		== The Big Picture ==
	* Two stretches of linear DNA with homologous ends can be combined using this technique.		* Two stretches of linear DNA with homologous ends can be combined using this technique.
-	* Useful ~~paper~~: [http://www.ncbi.nlm.nih.gov/pubmed/17446874 Gene splicing and mutagenesis by PCR-driven overlap extension]	+	* Useful papers:
		+	** [http://www.ncbi.nlm.nih.gov/pubmed/17446874 Gene splicing and mutagenesis by PCR-driven overlap extension]
		+	** For longer fragments: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373371/?tool=pmcentrez&rendertype=abstract Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously]

	== Obtain DNA ==		== Obtain DNA ==

Janet B. Matsen: /* Sewing PCR Without Primers */

2012-10-11T16:59:26Z

Sewing PCR Without Primers

←Older revision		Revision as of 16:59, 11 October 2012
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	== Sewing PCR Without Primers ==		== Sewing PCR Without Primers ==
	* use template DNA		* use template DNA
-	** ~~Concentration?~~	+	** Use ~ 50 - 125 ng of each piece per 50 uL. Each reaction should be 10 - 15 uL: do many so you can test a range of T<sub>anneal</sub> and % DMSO
	* Rxn concentrations		* Rxn concentrations
	** Use Phusion buffer & standard Phusion dNTP concentration		** Use Phusion buffer & standard Phusion dNTP concentration
-	** Set up an array of annealing temps and % DMSO.	+	** Set up an array of annealing temps and % DMSO to identify conditions that yield product and minimize unspecific bands. You can try 55<sup>o</sup>C, 60<sup>o</sup>C, 65<sup>o</sup>C, 70<sup>o</sup>C and 0, 5, & 10% DMSO at each of those temps to cover a very broad range of reaction conditions.
	*Thermocycling: Use Phusion polymerase with default concentrations.		*Thermocycling: Use Phusion polymerase with default concentrations.
	#98<sup>o</sup>C, 30sec		#98<sup>o</sup>C, 30sec
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	* You can try to run this product on a gel, but it is possible you won't see anything. (Depends on template DNA concentration in beginning.)		* You can try to run this product on a gel, but it is possible you won't see anything. (Depends on template DNA concentration in beginning.)
	* You can also do the next PCR step without examining on a gel.		* You can also do the next PCR step without examining on a gel.
-	** Janet recommends proceeding to this step unless you have problems. If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.	+	** Janet recommends proceeding to this step unless you have problems. If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.

	== Use Primers on "Sewing PCR" product ==		== Use Primers on "Sewing PCR" product ==

Janet B. Matsen at 16:56, 11 October 2012

2012-10-11T16:56:14Z

←Older revision		Revision as of 16:56, 11 October 2012
Line 7:		Line 7:
	== The Big Picture ==		== The Big Picture ==
	* Two stretches of linear DNA with homologous ends can be combined using this technique.		* Two stretches of linear DNA with homologous ends can be combined using this technique.
		+	* Useful paper: [http://www.ncbi.nlm.nih.gov/pubmed/17446874 Gene splicing and mutagenesis by PCR-driven overlap extension]

	== Obtain DNA ==		== Obtain DNA ==

Janet B. Matsen at 19:06, 10 October 2012

2012-10-10T19:06:58Z

←Older revision		Revision as of 19:06, 10 October 2012
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	This method can be used for cloning. It can also be used to assemble neighboring fragments in Gibson into one piece.		This method can be used for cloning. It can also be used to assemble neighboring fragments in Gibson into one piece.
		+
		+	== The Big Picture ==
		+	* Two stretches of linear DNA with homologous ends can be combined using this technique.

	== Obtain DNA ==		== Obtain DNA ==

Janet B. Matsen: /* Use Primers on Gel-Purified "Sewing PCR" product */

2012-10-02T17:15:40Z

Use Primers on Gel-Purified "Sewing PCR" product

←Older revision		Revision as of 17:15, 2 October 2012
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	** Janet recommends proceeding to this step unless you have problems. If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.		** Janet recommends proceeding to this step unless you have problems. If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.

-	== Use Primers on ~~Gel-Purified~~ "Sewing PCR" product ==	+	== Use Primers on "Sewing PCR" product ==
-	*	+	*optional: gel purify the product from the 1st rxn.
		+	** can run a few uL on a gel to see if non-specific products formed.

	== Extra Notes ==		== Extra Notes ==
	* Be aware that overloading an agarose gel leads to warped rates of migration through the gel. Include small not-overloaded lanes next to the ladder when running purification gels.		* Be aware that overloading an agarose gel leads to warped rates of migration through the gel. Include small not-overloaded lanes next to the ladder when running purification gels.
	[[image:janet purification gel.jpg\|thumb\|center\|include small not-overloaded lanes when running purification gels]]		[[image:janet purification gel.jpg\|thumb\|center\|include small not-overloaded lanes when running purification gels]]

Janet B. Matsen at 17:00, 2 October 2012

2012-10-02T17:00:11Z

←Older revision		Revision as of 17:00, 2 October 2012
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	#4<sup>o</sup>C, forever		#4<sup>o</sup>C, forever
	* You can try to run this product on a gel, but it is possible you won't see anything. (Depends on template DNA concentration in beginning.)		* You can try to run this product on a gel, but it is possible you won't see anything. (Depends on template DNA concentration in beginning.)
		+	* You can also do the next PCR step without examining on a gel.
		+	** Janet recommends proceeding to this step unless you have problems. If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.

	== Use Primers on Gel-Purified "Sewing PCR" product ==		== Use Primers on Gel-Purified "Sewing PCR" product ==
	*		*
		+
		+	== Extra Notes ==
		+	* Be aware that overloading an agarose gel leads to warped rates of migration through the gel. Include small not-overloaded lanes next to the ladder when running purification gels.
		+	[[image:janet purification gel.jpg\|thumb\|center\|include small not-overloaded lanes when running purification gels]]

Janet B. Matsen: /* Sewing PCR Without Primers */

2012-10-02T14:21:28Z

Sewing PCR Without Primers

←Older revision		Revision as of 14:21, 2 October 2012
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	#72<sup>o</sup>C, 5min		#72<sup>o</sup>C, 5min
	#4<sup>o</sup>C, forever		#4<sup>o</sup>C, forever
-	# You can try to run this product on a gel, but it is possible you won't see anything. (Depends on template DNA concentration in beginning.)	+	* You can try to run this product on a gel, but it is possible you won't see anything. (Depends on template DNA concentration in beginning.)

	== Use Primers on Gel-Purified "Sewing PCR" product ==		== Use Primers on Gel-Purified "Sewing PCR" product ==
	*		*

Janet B. Matsen: /* Sewing PCR Without Primers */

2012-10-02T14:21:19Z

Sewing PCR Without Primers

←Older revision		Revision as of 14:21, 2 October 2012
Line 25:		Line 25:
	#72<sup>o</sup>C, 5min		#72<sup>o</sup>C, 5min
	#4<sup>o</sup>C, forever		#4<sup>o</sup>C, forever
		+	# You can try to run this product on a gel, but it is possible you won't see anything. (Depends on template DNA concentration in beginning.)

	== Use Primers on Gel-Purified "Sewing PCR" product ==		== Use Primers on Gel-Purified "Sewing PCR" product ==
	*		*