Site-directed mutagenesis - Revision history

Fergal O'Meara at 16:53, 9 May 2012

2012-05-09T16:53:11Z

←Older revision		Revision as of 16:53, 9 May 2012
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	*For multiple non-adjacent mutations, you can use a method that incorporates PCR and Type IIs restriction systems to incorporate multiple mutations <cite>ko-ma</cite>. --[[Sri Kosuri]] ([[User talk:Skosuri\|talk]]) 15:27, 10 November 2006 (EST)		*For multiple non-adjacent mutations, you can use a method that incorporates PCR and Type IIs restriction systems to incorporate multiple mutations <cite>ko-ma</cite>. --[[Sri Kosuri]] ([[User talk:Skosuri\|talk]]) 15:27, 10 November 2006 (EST)
	*Note that the primer design in the protocol of Stratagen is not very perfect. Cause it has a limitation on the Tm of the primers.Here I found a good reference which shows a better method to design the primers and expands the use of QCM. Learn more: http://nar.oxfordjournals.org/cgi/content/full/32/14/e115		*Note that the primer design in the protocol of Stratagen is not very perfect. Cause it has a limitation on the Tm of the primers.Here I found a good reference which shows a better method to design the primers and expands the use of QCM. Learn more: http://nar.oxfordjournals.org/cgi/content/full/32/14/e115
-		+	*I've found that the method is very sensitive to the reagents. If you are having trouble, and are confident its not a problem with primer sequence, try using fresh reagents for everything; polymerase, buffer, dntps (never freeze thaw more than once), primers (same re freezing), competent cells. Just because your reagents work on a standard PCR or transformation doesn't mean that they are not the problem in your QuikChange rxn. Presumably this is because (as already pointed out above) QuikChange is not a PCR method i.e. where product increases exponentially. I've found that the most robust components are the template plasmid (froze/thawed same aliquot for 4 years!) and dpn1. Another tip is to use a PCR clean up kit after the Dpn1 digest step (this removes traces of detergeant from the PCR buffer which the cells are extremely sensitive to), elute with 30ul and then add 7-8ul of this to 50ul competent cells. - [[User:Fergal O'Meara\|Fergal]]
	==References==		==References==

Christopher C Vanlang: /* Manuals */

2011-12-29T01:12:17Z

Manuals

←Older revision		Revision as of 01:12, 29 December 2011
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	===Manuals===		===Manuals===
-	*[http://www.~~stratagene~~.com/~~manuals~~/~~200518~~.pdf Stratagene QuikChange Site-directed Mutagenesis Kit]	+	*[http://www.genomics.agilent.com/files/Manual/200521.pdf Stratagene QuikChange Site-directed Mutagenesis Kit]

	===Publications===		===Publications===

Christopher C Vanlang: /* References */

2011-12-29T01:09:42Z

References

←Older revision		Revision as of 01:09, 29 December 2011
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	</biblio>		</biblio>

		+	===See Also===
		+	*http://www.bioinformatics.org/primerx/

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:DNA]]		[[Category:DNA]]
	[[Category:In vitro]]		[[Category:In vitro]]

Michael A. Speer: /* Specific Protocols */

2011-01-25T21:54:24Z

Specific Protocols

←Older revision		Revision as of 21:54, 25 January 2011
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	*[[Endy:Site-directed Mutagenesis]]		*[[Endy:Site-directed Mutagenesis]]
	*[[Knight:Site-directed mutagenesis]]		*[[Knight:Site-directed mutagenesis]]
-	*[[Richard Lab:Site Directed Mutagenesis]]	+	*[[Richard Lab:Site Directed Mutagenesis\|Richard Lab:Site-directed Mutagenesis]]
	*[['Round-the-horn site-directed mutagenesis]]		*[['Round-the-horn site-directed mutagenesis]]
	*[[Overlap Extension PCR]]		*[[Overlap Extension PCR]]

Michael A. Speer: /* Specific Protocols */

2011-01-25T21:53:25Z

Specific Protocols

←Older revision		Revision as of 21:53, 25 January 2011
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	*[[Endy:Site-directed Mutagenesis]]		*[[Endy:Site-directed Mutagenesis]]
	*[[Knight:Site-directed mutagenesis]]		*[[Knight:Site-directed mutagenesis]]
		+	*[[Richard Lab:Site Directed Mutagenesis]]
	*[['Round-the-horn site-directed mutagenesis]]		*[['Round-the-horn site-directed mutagenesis]]
	*[[Overlap Extension PCR]]		*[[Overlap Extension PCR]]

Torsten Waldminghaus at 10:43, 19 February 2009

2009-02-19T10:43:43Z

←Older revision		Revision as of 10:43, 19 February 2009
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	#Purify template plasmid DNA from a dam<sup>+</sup> ''Escherichia coli'' strain (to ensure that all GATC sites are methylated for later digestion with DpnI).		#Purify template plasmid DNA from a dam<sup>+</sup> ''Escherichia coli'' strain (to ensure that all GATC sites are methylated for later digestion with DpnI).
	#Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make. See the CAD tool [http://www.bioinformatics.org/primerx/documentation.html PrimerX].		#Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make. See the CAD tool [http://www.bioinformatics.org/primerx/documentation.html PrimerX].
-	#Run a primer-extension reaction with a proof-reading, non-displacing polymerase such as ''~~PFU~~'' DNA polymerase. This results in nicked circular strands of the plasmid.	+	#Run a primer-extension reaction with a proof-reading, non-displacing polymerase such as ''[[Pfu]]'' DNA polymerase. This results in nicked circular strands of the plasmid.
	#Cut up the template DNA with DpnI.		#Cut up the template DNA with DpnI.
	#Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks and not restrict the unmodified product DNA.		#Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks and not restrict the unmodified product DNA.

Tk: /* General Procedure */

2008-11-24T18:58:01Z

General Procedure

←Older revision		Revision as of 18:58, 24 November 2008
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	==General Procedure==		==General Procedure==
	#Purify template plasmid DNA from a dam<sup>+</sup> ''Escherichia coli'' strain (to ensure that all GATC sites are methylated for later digestion with DpnI).		#Purify template plasmid DNA from a dam<sup>+</sup> ''Escherichia coli'' strain (to ensure that all GATC sites are methylated for later digestion with DpnI).
-	#Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make.	+	#Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make. See the CAD tool [http://www.bioinformatics.org/primerx/documentation.html PrimerX].
	#Run a primer-extension reaction with a proof-reading, non-displacing polymerase such as ''PFU'' DNA polymerase. This results in nicked circular strands of the plasmid.		#Run a primer-extension reaction with a proof-reading, non-displacing polymerase such as ''PFU'' DNA polymerase. This results in nicked circular strands of the plasmid.
	#Cut up the template DNA with DpnI.		#Cut up the template DNA with DpnI.

Charles Liang: /* Notes */

2008-09-10T09:29:46Z

Notes

←Older revision		Revision as of 09:29, 10 September 2008
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	*For enhanced amplification try using the primer design from this paper<cite>Zheng-NAR-2004</cite>. To reduce self-primer annealing it uses oligos that do not completely overlap. I've made up to 60bp deletions with QuickChange, but it can be really, really painful. Point mutants are much easier. -[[User:Icb410\|Ian]]		*For enhanced amplification try using the primer design from this paper<cite>Zheng-NAR-2004</cite>. To reduce self-primer annealing it uses oligos that do not completely overlap. I've made up to 60bp deletions with QuickChange, but it can be really, really painful. Point mutants are much easier. -[[User:Icb410\|Ian]]
	*For multiple non-adjacent mutations, you can use a method that incorporates PCR and Type IIs restriction systems to incorporate multiple mutations <cite>ko-ma</cite>. --[[Sri Kosuri]] ([[User talk:Skosuri\|talk]]) 15:27, 10 November 2006 (EST)		*For multiple non-adjacent mutations, you can use a method that incorporates PCR and Type IIs restriction systems to incorporate multiple mutations <cite>ko-ma</cite>. --[[Sri Kosuri]] ([[User talk:Skosuri\|talk]]) 15:27, 10 November 2006 (EST)
-	*~~[[User:king6731253@gmail.com\|Liang]]~~Note that the primer design in the protocol of Stratagen is not very perfect. Cause it has a limitation on the Tm of the primers.Here I found a good reference which shows a better method to design the primers and expands the use of QCM. Learn more: http://nar.oxfordjournals.org/cgi/content/full/32/14/e115	+	*Note that the primer design in the protocol of Stratagen is not very perfect. Cause it has a limitation on the Tm of the primers.Here I found a good reference which shows a better method to design the primers and expands the use of QCM. Learn more: http://nar.oxfordjournals.org/cgi/content/full/32/14/e115

	==References==		==References==

Charles Liang: /* Notes */

2008-09-10T09:29:09Z

Notes

←Older revision		Revision as of 09:29, 10 September 2008
Line 31:		Line 31:
	*For enhanced amplification try using the primer design from this paper<cite>Zheng-NAR-2004</cite>. To reduce self-primer annealing it uses oligos that do not completely overlap. I've made up to 60bp deletions with QuickChange, but it can be really, really painful. Point mutants are much easier. -[[User:Icb410\|Ian]]		*For enhanced amplification try using the primer design from this paper<cite>Zheng-NAR-2004</cite>. To reduce self-primer annealing it uses oligos that do not completely overlap. I've made up to 60bp deletions with QuickChange, but it can be really, really painful. Point mutants are much easier. -[[User:Icb410\|Ian]]
	*For multiple non-adjacent mutations, you can use a method that incorporates PCR and Type IIs restriction systems to incorporate multiple mutations <cite>ko-ma</cite>. --[[Sri Kosuri]] ([[User talk:Skosuri\|talk]]) 15:27, 10 November 2006 (EST)		*For multiple non-adjacent mutations, you can use a method that incorporates PCR and Type IIs restriction systems to incorporate multiple mutations <cite>ko-ma</cite>. --[[Sri Kosuri]] ([[User talk:Skosuri\|talk]]) 15:27, 10 November 2006 (EST)
-	*[[User:~~Yuanjin~~\|Liang]]Note that the primer design in the protocol of Stratagen is not very perfect. Cause it has a limitation on the Tm of the primers.Here I found a good reference which shows a better method to design the primers and expands the use of QCM. Learn more: http://nar.oxfordjournals.org/cgi/content/full/32/14/e115	+	*[[User:king6731253@gmail.com\|Liang]]Note that the primer design in the protocol of Stratagen is not very perfect. Cause it has a limitation on the Tm of the primers.Here I found a good reference which shows a better method to design the primers and expands the use of QCM. Learn more: http://nar.oxfordjournals.org/cgi/content/full/32/14/e115

	==References==		==References==

Charles Liang: /* Notes */

2008-09-10T09:28:24Z

Notes

←Older revision		Revision as of 09:28, 10 September 2008
Line 31:		Line 31:
	*For enhanced amplification try using the primer design from this paper<cite>Zheng-NAR-2004</cite>. To reduce self-primer annealing it uses oligos that do not completely overlap. I've made up to 60bp deletions with QuickChange, but it can be really, really painful. Point mutants are much easier. -[[User:Icb410\|Ian]]		*For enhanced amplification try using the primer design from this paper<cite>Zheng-NAR-2004</cite>. To reduce self-primer annealing it uses oligos that do not completely overlap. I've made up to 60bp deletions with QuickChange, but it can be really, really painful. Point mutants are much easier. -[[User:Icb410\|Ian]]
	*For multiple non-adjacent mutations, you can use a method that incorporates PCR and Type IIs restriction systems to incorporate multiple mutations <cite>ko-ma</cite>. --[[Sri Kosuri]] ([[User talk:Skosuri\|talk]]) 15:27, 10 November 2006 (EST)		*For multiple non-adjacent mutations, you can use a method that incorporates PCR and Type IIs restriction systems to incorporate multiple mutations <cite>ko-ma</cite>. --[[Sri Kosuri]] ([[User talk:Skosuri\|talk]]) 15:27, 10 November 2006 (EST)
-	*Note that the primer design in the protocol of Stratagen is not very perfect. Cause it has a limitation on the ~~T~~m~~~~ of the primers.Here I found a good reference which shows a better method to design the primers and expands the use of QCM. Learn more: http://nar.oxfordjournals.org/cgi/content/full/32/14/e115	+	*[[User:Yuanjin\|Liang]]Note that the primer design in the protocol of Stratagen is not very perfect. Cause it has a limitation on the Tm of the primers.Here I found a good reference which shows a better method to design the primers and expands the use of QCM. Learn more: http://nar.oxfordjournals.org/cgi/content/full/32/14/e115

	==References==		==References==