Knight:Site-directed mutagenesis/Single site - Revision history

Tk: /* Procedure */

Tk — Mon, 24 Nov 2008 18:59:59 GMT

Procedure

←Older revision		Revision as of 18:59, 24 November 2008
Line 26:		Line 26:

	#Design mutagenesis primers.		#Design mutagenesis primers.
-	#*The primer should be designed so that the desired mutation occurs at the exact center of the primer. The forward and reverse primers should be complementary to each other (i.e. anneal to the same location on opposite strands of the template).	+	#*The primer should be designed so that the desired mutation occurs at the exact center of the primer. The forward and reverse primers should be complementary to each other (i.e. anneal to the same location on opposite strands of the template). See the CAD tool [http://www.bioinformatics.org/primerx/documentation.html PrimerX]
	#*Aim for ~15 or more bases of identical flanking sequence on each side of the mutation. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure.		#*Aim for ~15 or more bases of identical flanking sequence on each side of the mutation. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure.
	#*Stratagene strongly recommends using FPLC or PAGE purified primers. See [[Designing primers#Oligo synthesis information \| here]] for links to information on oligo purification. 5' phosphorylation is not necessary.		#*Stratagene strongly recommends using FPLC or PAGE purified primers. See [[Designing primers#Oligo synthesis information \| here]] for links to information on oligo purification. 5' phosphorylation is not necessary.

Reshma P. Shetty: Knight:Site-directed mutagenesis moved to Knight:Site-directed mutagenesis/Single site: adding a new protocol for multi site mutagenesis

Reshma P. Shetty — Thu, 23 Aug 2007 19:38:38 GMT

Knight:Site-directed mutagenesis moved to Knight:Site-directed mutagenesis/Single site: adding a new protocol for multi site mutagenesis

←Older revision

Revision as of 19:38, 23 August 2007

Reshma P. Shetty at 17:37, 2 June 2007

Reshma P. Shetty — Sat, 02 Jun 2007 17:37:51 GMT

←Older revision		Revision as of 17:37, 2 June 2007
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	#[[Knight:Electroporation \| Transform]] purified DNA into highly competent cells.		#[[Knight:Electroporation \| Transform]] purified DNA into highly competent cells.
	#Screen the transformants for the desired mutation using [[Colony PCR \| colony PCR]], [[Restriction Digest \| restriction digest]] or [[Sequencing DNA \| sequencing]] as appropriate. Typically 1/4 or 1/8 colonies are correct.		#Screen the transformants for the desired mutation using [[Colony PCR \| colony PCR]], [[Restriction Digest \| restriction digest]] or [[Sequencing DNA \| sequencing]] as appropriate. Typically 1/4 or 1/8 colonies are correct.
		+
		+	[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]]

Reshma P. Shetty: /* Mutagenesis PCR mix */

Reshma P. Shetty — Mon, 12 Feb 2007 02:35:52 GMT

Mutagenesis PCR mix

←Older revision		Revision as of 02:35, 12 February 2007
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	==Mutagenesis PCR mix==		==Mutagenesis PCR mix==
-	*5 μL of 10X reaction buffer	+	*5 μL of 10X reaction buffer (comes with PfuTurbo DNA polymerase)
	*X μL (5–50 ng) of dsDNA template		*X μL (5–50 ng) of dsDNA template
	*X μL (125 ng) of oligonucleotide primer #1		*X μL (125 ng) of oligonucleotide primer #1
	*X μL (125 ng) of oligonucleotide primer #2		*X μL (125 ng) of oligonucleotide primer #2
-	*1 μL of dNTP mix (~~usually 25 mM stock of each dNTP so~~ 100mM total dNTP mix)	+	*1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)
	*ddH<sub>2</sub>O to a final volume of 50 μL		*ddH<sub>2</sub>O to a final volume of 50 μL
	Then add		Then add
	*1 μL of PfuTurbo DNA polymerase (2.5 U/μL)		*1 μL of PfuTurbo DNA polymerase (2.5 U/μL)
-		+
	==Procedure==		==Procedure==
	This procedure is primarily derived from the [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Site-Directed Mutagenesis manual] with some modifications based on past experience.		This procedure is primarily derived from the [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Site-Directed Mutagenesis manual] with some modifications based on past experience.

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Wed, 16 Aug 2006 21:41:14 GMT

Procedure

←Older revision		Revision as of 21:41, 16 August 2006
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	##55°C for 1 min		##55°C for 1 min
	##68°C for 1 min/kb of plasmid length minimum		##68°C for 1 min/kb of plasmid length minimum
-	#*Run PCR for 18 cycles for point mutations (even though the Stratagene manual recommends 12, I've found 18 to be preferable).	+	##Run PCR for 18 cycles for point mutations (even though the Stratagene manual recommends 12, I've found 18 to be preferable).
		+	##68°C for 20 mins
	#*Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.		#*Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
	#*Note that it is pretty common to not be able to visualize the PCR product on a gel but yet have the mutagenesis still work.		#*Note that it is pretty common to not be able to visualize the PCR product on a gel but yet have the mutagenesis still work.

Jason R. Kelly: /* Procedure */

Jason R. Kelly — Tue, 18 Jul 2006 20:10:32 GMT

Procedure

←Older revision		Revision as of 20:10, 18 July 2006
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	#Purify PCR product.		#Purify PCR product.
	#*I typically do this step using a [http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf QIAgen PCR Purification kit] but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.		#*I typically do this step using a [http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf QIAgen PCR Purification kit] but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.
-	#[[Electroporation \| Transform]] purified DNA into highly competent cells.	+	#[[Knight:Electroporation \| Transform]] purified DNA into highly competent cells.
	#Screen the transformants for the desired mutation using [[Colony PCR \| colony PCR]], [[Restriction Digest \| restriction digest]] or [[Sequencing DNA \| sequencing]] as appropriate. Typically 1/4 or 1/8 colonies are correct.		#Screen the transformants for the desired mutation using [[Colony PCR \| colony PCR]], [[Restriction Digest \| restriction digest]] or [[Sequencing DNA \| sequencing]] as appropriate. Typically 1/4 or 1/8 colonies are correct.

Tk: /* Procedure */

Tk — Sun, 02 Jul 2006 21:03:42 GMT

Procedure

←Older revision		Revision as of 21:03, 2 July 2006
Line 26:		Line 26:

	#Design mutagenesis primers.		#Design mutagenesis primers.
-	#*The primer should be designed so that the desired mutation occurs at the exact center of the primer. The forward and reverse primers should be complementary to ~~eachother~~ (i.e. anneal to the same location on opposite strands of the template).	+	#*The primer should be designed so that the desired mutation occurs at the exact center of the primer. The forward and reverse primers should be complementary to each other (i.e. anneal to the same location on opposite strands of the template).
	#*Aim for ~15 or more bases of identical flanking sequence on each side of the mutation. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure.		#*Aim for ~15 or more bases of identical flanking sequence on each side of the mutation. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure.
	#*Stratagene strongly recommends using FPLC or PAGE purified primers. See [[Designing primers#Oligo synthesis information \| here]] for links to information on oligo purification. 5' phosphorylation is not necessary.		#*Stratagene strongly recommends using FPLC or PAGE purified primers. See [[Designing primers#Oligo synthesis information \| here]] for links to information on oligo purification. 5' phosphorylation is not necessary.
Line 46:		Line 46:
	#Purify PCR product.		#Purify PCR product.
	#*I typically do this step using a [http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf QIAgen PCR Purification kit] but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.		#*I typically do this step using a [http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf QIAgen PCR Purification kit] but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.
-	#[[Electroporation \| ~~Tranform~~]] purified DNA into highly competent cells.	+	#[[Electroporation \| Transform]] purified DNA into highly competent cells.
	#Screen the transformants for the desired mutation using [[Colony PCR \| colony PCR]], [[Restriction Digest \| restriction digest]] or [[Sequencing DNA \| sequencing]] as appropriate. Typically 1/4 or 1/8 colonies are correct.		#Screen the transformants for the desired mutation using [[Colony PCR \| colony PCR]], [[Restriction Digest \| restriction digest]] or [[Sequencing DNA \| sequencing]] as appropriate. Typically 1/4 or 1/8 colonies are correct.

Tk: /* Materials */

Tk — Sun, 02 Jul 2006 21:02:04 GMT

Materials

←Older revision		Revision as of 21:02, 2 July 2006
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	*PCR Thermocycler		*PCR Thermocycler
	*[http://www.neb.com/nebecomm/products/productR0176.asp Dpn I]		*[http://www.neb.com/nebecomm/products/productR0176.asp Dpn I]
-	*~~Compentent~~ cells	+	*Competent cells

	==Mutagenesis PCR mix==		==Mutagenesis PCR mix==

Reshma P. Shetty at 12:49, 24 August 2005

Reshma P. Shetty — Wed, 24 Aug 2005 12:49:14 GMT

New page

''Back to [[Site-directed mutagenesis]]''

Note that this is a work in progress and needs to be verified against my lab notebooks. --[[User:Rshetty|Reshma]] 08:49, 24 Aug 2005 (EDT)

==Materials==
*Template plasmid purified from a dam+ ''E. coli'' strain
*Mutatagenesis primers
*[http://www.stratagene.com/products/showProduct.aspx?pid=105 ''PfuTurbo'' DNA polymerase] (nonstrand-displacing) and associated reaction buffer
*dNTPs
*PCR Thermocycler
*[http://www.neb.com/nebecomm/products/productR0176.asp Dpn I]
*Compentent cells

==Mutagenesis PCR mix==
*5 μL of 10X reaction buffer
*X μL (5–50 ng) of dsDNA template
*X μL (125 ng) of oligonucleotide primer #1
*X μL (125 ng) of oligonucleotide primer #2
*1 μL of dNTP mix (usually 25 mM stock of each dNTP so 100mM total dNTP mix)
*ddH2O to a final volume of 50 μL
Then add
*1 μL of PfuTurbo DNA polymerase (2.5 U/μL)

==Procedure==
This procedure is primarily derived from the [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Site-Directed Mutagenesis manual] with some modifications based on past experience.

#Design mutagenesis primers.
#*The primer should be designed so that the desired mutation occurs at the exact center of the primer. The forward and reverse primers should be complementary to eachother (i.e. anneal to the same location on opposite strands of the template).
#*Aim for ~15 or more bases of identical flanking sequence on each side of the mutation. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure.
#*Stratagene strongly recommends using FPLC or PAGE purified primers. See [[Designing primers#Oligo synthesis information | here]] for links to information on oligo purification. 5' phosphorylation is not necessary.
#*See the [http://www.stratagene.com/manuals/200518.pdf Stratagene manual] for more detailed information. In particular, adhere to their formula for calculating the melting temperature of your primers and design your primers to have a melting temperature >=78°C.
#*See [[Designing primers | designing primers]] for general advice on primer design.
#Purify template plasmid from a dam+ ''E. coli'' strain via [[Miniprep | miniprep]].
#Set up mutagenesis PCR mix as described above.
#Run PCR
##95°C for 2 mins
##95°C for 30 secs
##55°C for 1 min
##68°C for 1 min/kb of plasmid length minimum
#*Run PCR for 18 cycles for point mutations (even though the Stratagene manual recommends 12, I've found 18 to be preferable).
#*Stratagene recommends using a PCR machine with heated lid or overlaying the reaction with mineral oil.
#*Note that it is pretty common to not be able to visualize the PCR product on a gel but yet have the mutagenesis still work.
#Cool the reaction to <=37°C
#Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).
#Incubate 2-3 hours at 37°C (even though the Stratagene manual only recommends 1 hr).
#Purify PCR product.
#*I typically do this step using a [http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf QIAgen PCR Purification kit] but any purification which removes the salts, dNTPs, oligos and proteins from the PCR should be fine.
#[[Electroporation | Tranform]] purified DNA into highly competent cells.
#Screen the transformants for the desired mutation using [[Colony PCR | colony PCR]], [[Restriction Digest | restriction digest]] or [[Sequencing DNA | sequencing]] as appropriate. Typically 1/4 or 1/8 colonies are correct.