'Round-the-horn site-directed mutagenesis - Revision history

Etienne Robillard: /* Background */

Etienne Robillard — Thu, 28 Mar 2013 11:37:01 GMT

Background

←Older revision		Revision as of 11:37, 28 March 2013
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-	'Round-the-horn is a PCR-based mutagenesis. The ~~muations~~ are contained in one or both of the primers. The primers are phosphorylated so that the PCR product can be ligated into a circle and used to transform cells. The procedure has the advantages that the primers are smaller, a visible product on a gel means your reaction worked and must contain the encoded mutations, only one primer needs to be changed to change the mutation at the same site. If you are concerned with mutations elsewhere in your plasmid, you should sub-clone the segment of interest into a "fresh" vector (this is the case for any site-directed protocol). Finally, as opposed to standard site-directed mutagenesis, this procedure exponentially amplifies the plasmid, leading to increased yields, increased transformation efficiencies, and the potential to create a large library with randomized residues.	+	'Round-the-horn is a PCR-based mutagenesis. The mutations are contained in one or both of the primers. The primers are phosphorylated so that the PCR product can be ligated into a circle and used to transform cells. The procedure has the advantages that the primers are smaller, a visible product on a gel means your reaction worked and must contain the encoded mutations, only one primer needs to be changed to change the mutation at the same site. If you are concerned with mutations elsewhere in your plasmid, you should sub-clone the segment of interest into a "fresh" vector (this is the case for any site-directed protocol). Finally, as opposed to standard site-directed mutagenesis, this procedure exponentially amplifies the plasmid, leading to increased yields, increased transformation efficiencies, and the potential to create a large library with randomized residues.

	[[Image:SDMHorn1a.tif\|center\|Horn Schematic]]		[[Image:SDMHorn1a.tif\|center\|Horn Schematic]]

Smoore: /* Optimizations */

Smoore — Fri, 31 Aug 2012 13:07:14 GMT

Optimizations

←Older revision		Revision as of 13:07, 31 August 2012
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	Keep in mind that when the sample temperature is raised from the annealing temperature (say 55) to the optimal extension temperature (say 72), the oligo would normally melt off of the template. The reason primer extension works is the polymerase slowly extends the oligo during annealing and while the temperature is rising so that when the 72 degree mark is hit, the extended product is long enough to keep the primer attached and the polymerase happy. Therefore, oligo composition, the type of polymerase, the annealing temp, and ramp time can have significant effects on the reaction.		Keep in mind that when the sample temperature is raised from the annealing temperature (say 55) to the optimal extension temperature (say 72), the oligo would normally melt off of the template. The reason primer extension works is the polymerase slowly extends the oligo during annealing and while the temperature is rising so that when the 72 degree mark is hit, the extended product is long enough to keep the primer attached and the polymerase happy. Therefore, oligo composition, the type of polymerase, the annealing temp, and ramp time can have significant effects on the reaction.

-	Take the following example: Each reaction had 1 μL of the same mini-prep, and all reactions were done with the same program at the same time. Three different primer pairs were used and three different enzymes. I have found that the surest way to get a successful 'Horn reaction is to set up the reactions with different enzymes (~~usually~~ Deep Vent and Turbo Pfu) rather than to set up arrays of Mg<sup>++</sup> concentration or a gradient of annealing temps. The desired product is marked with an arrow. Note that the different enzymes gave varied performance simply depending on the primer pair used.	+	Take the following example: Each reaction had 1 μL of the same mini-prep, and all reactions were done with the same program at the same time. Three different primer pairs were used and three different enzymes. I have found that the surest way to get a successful 'Horn reaction is to set up the reactions with different enzymes (historically Deep Vent and Turbo Pfu, but now an array of fusion enzymes) rather than to set up arrays of Mg<sup>++</sup> concentration or a gradient of annealing temps. The desired product is marked with an arrow. Note that the different enzymes gave varied performance simply depending on the primer pair used. Consider the cost of your time repeating steps relative to the cost of the enzyme aliquot.

	[[Image:SDMHorn2a.tif\|center\|Example Horn Gel]]		[[Image:SDMHorn2a.tif\|center\|Example Horn Gel]]

Smoore: /* PCR program */

Smoore — Fri, 31 Aug 2012 13:04:53 GMT

PCR program

←Older revision		Revision as of 13:04, 31 August 2012
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	# 98°C 1’		# 98°C 1’
	# 96°C 30”		# 96°C 30”
-	# 55-58°C 30” (~5°C below T<sub>m</sub>)	+	# 55-58°C 30” (~5°C below T<sub>m</sub>, higher for fusion enzymes)
	# 72°C 2’ per kb being amplified (much less if using a fusion enzyme)		# 72°C 2’ per kb being amplified (much less if using a fusion enzyme)
	# Goto (2) 25-27 more times		# Goto (2) 25-27 more times
-	# cool to 18°C for a minute or so	+	# cool to 18°C for a minute or so (there's no need to hold at 4, you just cooked the sample for an hour!)
	# end		# end

Smoore: /* The Mixture */

Smoore — Fri, 31 Aug 2012 12:55:52 GMT

The Mixture

←Older revision		Revision as of 12:55, 31 August 2012
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	===The Mixture===		===The Mixture===
-	Set up the following 50μL mixture on ice. After mixing, put into pre-heated PCR block.	+	Set up the following 50μL mixture on ice. After mixing, put into pre-heated PCR block. Keep in mind that your particular volumes may be different, some newer fusion polymerases (recommended) use 5X and 2X buffers.

	*40 μL water		*40 μL water

Oscar Weisberg at 20:46, 29 August 2012

Oscar Weisberg — Wed, 29 Aug 2012 20:46:34 GMT

←Older revision		Revision as of 20:46, 29 August 2012
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	Set up the following 50μL mixture on ice. After mixing, put into pre-heated PCR block.		Set up the following 50μL mixture on ice. After mixing, put into pre-heated PCR block.

-	*39 μL water	+	*40 μL water
	*5 μL 10X polymerase buffer		*5 μL 10X polymerase buffer
	*1.5 μL forward primer (0.3 μM final)		*1.5 μL forward primer (0.3 μM final)

Smoore: /* PCR program */

Smoore — Thu, 30 Sep 2010 13:51:39 GMT

PCR program

←Older revision		Revision as of 13:51, 30 September 2010
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	===PCR program===		===PCR program===

-	# ~~95°C~~ 1’	+	# 98°C 1’
-	# ~~93°C~~ 30”	+	# 96°C 30”
-	# ~~55°C~~ 30” (~5°C below T<sub>m</sub>)	+	# 55-58°C 30” (~5°C below T<sub>m</sub>)
-	# 72°C 2’ per kb being amplified	+	# 72°C 2’ per kb being amplified (much less if using a fusion enzyme)
-	# Goto (2) 25 more times	+	# Goto (2) 25-27 more times
-	# ~~4°C hold~~	+	# cool to 18°C for a minute or so
		+	# end

	==Product Preparation==		==Product Preparation==

Smoore: /* The Mixture */

Smoore — Thu, 30 Sep 2010 13:49:58 GMT

The Mixture

←Older revision		Revision as of 13:49, 30 September 2010
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	*1.5 μL forward primer (0.3 μM final)		*1.5 μL forward primer (0.3 μM final)
	*1.5 μL reverse primer		*1.5 μL reverse primer
-	*1 μL dNTPs (20 mM each)	+	*0.5 μL dNTPs (20 mM each)
	*1 μL template (mini-prep’d plasmid)		*1 μL template (mini-prep’d plasmid)
-	*1 μL polymerase (not Taq, use Vent, Deep Vent, 9°N, Pfu)	+	*0.5 μL polymerase (not Taq, use Vent, Deep Vent, 9°N, Pfu, or a faster fusion enzyme)

	===PCR program===		===PCR program===

Smoore: /* Background */

Smoore — Thu, 30 Sep 2010 13:48:56 GMT

Background

←Older revision		Revision as of 13:48, 30 September 2010
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-	'Round-the-horn is a PCR-based mutagenesis. The muations are contained in one or both of the primers. The primers are phosphorylated so that the PCR product can be ligated into a circle and used to transform cells. The procedure has the advantages that the primers are smaller, a visible product on a gel means your reaction worked and must contain the encoded mutations, only one primer needs to be changed to change the mutation at the same site. If you are concerned with mutations elsewhere in your plasmid, you should sub-clone the segment of interest into a "fresh" vector (this is the case for any site-directed protocol). Finally, as opposed to standard site-directed mutagenesis, this procedure exponentially amplifies the plasmid, leading to increased yields ~~and therefore~~ transformation efficiencies.	+	'Round-the-horn is a PCR-based mutagenesis. The muations are contained in one or both of the primers. The primers are phosphorylated so that the PCR product can be ligated into a circle and used to transform cells. The procedure has the advantages that the primers are smaller, a visible product on a gel means your reaction worked and must contain the encoded mutations, only one primer needs to be changed to change the mutation at the same site. If you are concerned with mutations elsewhere in your plasmid, you should sub-clone the segment of interest into a "fresh" vector (this is the case for any site-directed protocol). Finally, as opposed to standard site-directed mutagenesis, this procedure exponentially amplifies the plasmid, leading to increased yields, increased transformation efficiencies, and the potential to create a large library with randomized residues.

	[[Image:SDMHorn1a.tif\|center\|Horn Schematic]]		[[Image:SDMHorn1a.tif\|center\|Horn Schematic]]

Torsten Waldminghaus at 17:30, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 17:30:38 GMT

←Older revision		Revision as of 17:30, 23 February 2009
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		+	{{back to protocols}}
	‘Round the Horn Site-directed mutagenesis		‘Round the Horn Site-directed mutagenesis

Smoore: /* Optimizations */

Smoore — Thu, 03 Jul 2008 15:33:52 GMT

Optimizations

←Older revision		Revision as of 15:33, 3 July 2008
Line 108:		Line 108:
	Take the following example: Each reaction had 1 μL of the same mini-prep, and all reactions were done with the same program at the same time. Three different primer pairs were used and three different enzymes. I have found that the surest way to get a successful 'Horn reaction is to set up the reactions with different enzymes (usually Deep Vent and Turbo Pfu) rather than to set up arrays of Mg<sup>++</sup> concentration or a gradient of annealing temps. The desired product is marked with an arrow. Note that the different enzymes gave varied performance simply depending on the primer pair used.		Take the following example: Each reaction had 1 μL of the same mini-prep, and all reactions were done with the same program at the same time. Three different primer pairs were used and three different enzymes. I have found that the surest way to get a successful 'Horn reaction is to set up the reactions with different enzymes (usually Deep Vent and Turbo Pfu) rather than to set up arrays of Mg<sup>++</sup> concentration or a gradient of annealing temps. The desired product is marked with an arrow. Note that the different enzymes gave varied performance simply depending on the primer pair used.

-	[[~~image~~:SDMHorn2a.tif\|center\|Example Horn Gel]]	+	[[Image:SDMHorn2a.tif\|center\|Example Horn Gel]]