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

Austin J. Che: /* Procedure */

2007-08-23T20:25:46Z

Procedure

←Older revision		Revision as of 20:25, 23 August 2007
Line 51:		Line 51:
	##95°C for 1 min		##95°C for 1 min
	##55°C for 1 min		##55°C for 1 min
-	##65°C for 2 min/kb of plasmid length minimum	+	##65°C for 2 min/kb of plasmid length minimum (is [http://www.neb.com/nebecomm/products/faqproductM0208.asp#361 optimal] temperature for Taq ligase)
	##Run reaction for 30 cycles.		##Run reaction for 30 cycles.
	#*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.

Austin J. Che at 20:23, 23 August 2007

2007-08-23T20:23:45Z

←Older revision		Revision as of 20:23, 23 August 2007
Line 9:		Line 9:
	*Mutatagenesis primers (one per mutation, each on the same strand)		*Mutatagenesis primers (one per mutation, each on the same strand)
	*[http://www.stratagene.com/products/showProduct.aspx?pid=105 ''PfuTurbo'' DNA polymerase] (nonstrand-displacing) and associated reaction buffer		*[http://www.stratagene.com/products/showProduct.aspx?pid=105 ''PfuTurbo'' DNA polymerase] (nonstrand-displacing) and associated reaction buffer
-	*Taq ligase	+	*[http://www.neb.com/nebecomm/products/productM0208.asp Taq ligase]
	*dNTPs		*dNTPs
	*ATP		*ATP

Austin J. Che: /* Notes */

2007-08-23T20:22:15Z

Notes

←Older revision		Revision as of 20:22, 23 August 2007
Line 65:		Line 65:
	*Stratagene does not recommend this protocol for insertions or deletions.		*Stratagene does not recommend this protocol for insertions or deletions.
	*Apparently there are no primer spacing-dependent effects on mutagenesis efficiency (primers can be adjacent or far apart).		*Apparently there are no primer spacing-dependent effects on mutagenesis efficiency (primers can be adjacent or far apart).
		+	*You can also phosphorylate the primers separately from the rest of the mutagenesis reaction

	==Reference==		==Reference==

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

2007-08-23T20:14:55Z

Mutagenesis PCR mix

←Older revision		Revision as of 20:14, 23 August 2007
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	**For 1-3 primers, add 100 ng each primer. For 4-5 primers, add 50 ng each primer.		**For 1-3 primers, add 100 ng each primer. For 4-5 primers, add 50 ng each primer.
	**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.		**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.
		+	**Austin uses 0.1μL of 40μM primer (each one).
	*1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)		*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 22 μL		*ddH<sub>2</sub>O to a final volume of 22 μL

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

2007-08-23T20:14:00Z

Mutagenesis PCR mix

←Older revision		Revision as of 20:14, 23 August 2007
Line 27:		Line 27:
	**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.		**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.
	*1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)		*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 25 μL	+	*ddH<sub>2</sub>O to a final volume of 22 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)

Reshma P. Shetty: /* Procedure */

2007-08-23T20:13:43Z

Procedure

←Older revision		Revision as of 20:13, 23 August 2007
Line 55:		Line 55:
	#Cool the reaction to <=37°C		#Cool the reaction to <=37°C
	#Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).		#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).	+	#Incubate 6 hours at 37°C (even though the Stratagene manual only recommends 1 hr).
-	#Purify PCR product (not necessary, ~~I transform~~ 3 μl directly).	+	#Purify PCR product (not necessary, Austin transforms 3 μl directly).
	#*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.
	#[[Knight:Electroporation \| Transform]] purified DNA into highly competent cells.		#[[Knight:Electroporation \| Transform]] purified DNA into highly competent cells.

Austin J. Che at 20:09, 23 August 2007

2007-08-23T20:09:52Z

←Older revision		Revision as of 20:09, 23 August 2007
Line 11:		Line 11:
	*Taq ligase		*Taq ligase
	*dNTPs		*dNTPs
		+	*ATP
	*PCR Thermocycler		*PCR Thermocycler
	*[http://www.neb.com/nebecomm/products/productR0176.asp Dpn I]		*[http://www.neb.com/nebecomm/products/productR0176.asp Dpn I]
	*Competent cells		*Competent cells
		+	*unphosphorylated primers (1 for each mutation)

	==Mutagenesis PCR mix==		==Mutagenesis PCR mix==
-	*2.5 μL of 10X ~~reaction~~ buffer (~~comes with PfuTurbo DNA polymerase~~)	+	25μL total reaction volume:
-	*X μL (50 ng) of dsDNA template	+	*2.5 μL of 10X Taq ligase buffer (need the NAD for Taq ligase)
		+	*0.5 μL 100mM ATP
		+	*1 μL 25mM each dNTP
		+	*X μL (50-100 ng) of dsDNA template
	*X μL of each oligonucleotide primer		*X μL of each oligonucleotide primer
	**For 1-3 primers, add 100 ng each primer. For 4-5 primers, add 50 ng each primer.		**For 1-3 primers, add 100 ng each primer. For 4-5 primers, add 50 ng each primer.
Line 25:		Line 30:
	Then add		Then add
	*1 μL of PfuTurbo DNA polymerase (2.5 U/μL)		*1 μL of PfuTurbo DNA polymerase (2.5 U/μL)
-	*1 μL of Taq Ligase?	+	*1 μL of Taq Ligase
		+	*1 μL of T4 PNK

	==Procedure==		==Procedure==
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	#Purify template plasmid from a dam<sup>+</sup> ''E. coli'' strain via [[Miniprep \| miniprep]].		#Purify template plasmid from a dam<sup>+</sup> ''E. coli'' strain via [[Miniprep \| miniprep]].
	#Set up mutagenesis PCR mix as described above.		#Set up mutagenesis PCR mix as described above.
-	#Run ~~PCR~~	+	#Run Reaction
-	##95°C for 1 min	+	##37°C for 30 min (T4 PNK step)
		+	##95°C for 3 min
	##95°C for 1 min		##95°C for 1 min
	##55°C for 1 min		##55°C for 1 min
Line 49:		Line 56:
	#Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).		#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).		#Incubate 2-3 hours at 37°C (even though the Stratagene manual only recommends 1 hr).
-	#Purify PCR product.	+	#Purify PCR product (not necessary, I transform 3 μl directly).
	#*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.
	#[[Knight:Electroporation \| Transform]] purified DNA into highly competent cells.		#[[Knight:Electroporation \| Transform]] purified DNA into highly competent cells.

Reshma P. Shetty at 20:02, 23 August 2007

2007-08-23T20:02:26Z

←Older revision		Revision as of 20:02, 23 August 2007
Line 16:		Line 16:

	==Mutagenesis PCR mix==		==Mutagenesis PCR mix==
-	*5 μL of 10X reaction buffer (comes with PfuTurbo DNA polymerase)	+	*2.5 μL of 10X reaction buffer (comes with PfuTurbo DNA polymerase)
-	*X μL (~~5–50~~ ng) of dsDNA template	+	*X μL (50 ng) of dsDNA template
-	*X μL ~~(125 ng)~~ of each oligonucleotide primer	+	*X μL of each oligonucleotide primer
		+	**For 1-3 primers, add 100 ng each primer. For 4-5 primers, add 50 ng each primer.
	**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.		**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.
	*1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)		*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 25 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)
		+	*1 μL of Taq Ligase?

	==Procedure==		==Procedure==
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	#Set up mutagenesis PCR mix as described above.		#Set up mutagenesis PCR mix as described above.
	#Run PCR		#Run PCR
-	##95°C for ~~2 mins~~	+	##95°C for 1 min
-	##95°C for ~~30 secs~~	+	##95°C for 1 min
	##55°C for 1 min		##55°C for 1 min
-	##68°C for 1 min/kb of plasmid length minimum	+	##65°C for 2 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 reaction for 30 cycles.
-	~~##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.~~
	#Cool the reaction to <=37°C		#Cool the reaction to <=37°C
	#Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).		#Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage).
Line 52:		Line 52:
	#*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.
	#[[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.

	==Notes==		==Notes==

Reshma P. Shetty: in progress!

2007-08-23T19:57:45Z

in progress!

New page

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

Work in progress!

This protocol permits mutation at multiple sites simultaneously with only a single oligo per site.

==Materials==
*Template plasmid purified from a dam+ ''E. coli'' strain (not JM110 or SCS110)
*Mutatagenesis primers (one per mutation, each on the same strand)
*[http://www.stratagene.com/products/showProduct.aspx?pid=105 ''PfuTurbo'' DNA polymerase] (nonstrand-displacing) and associated reaction buffer
*Taq ligase
*dNTPs
*PCR Thermocycler
*[http://www.neb.com/nebecomm/products/productR0176.asp Dpn I]
*Competent cells

==Mutagenesis PCR mix==
*5 μL of 10X reaction buffer (comes with PfuTurbo DNA polymerase)
*X μL (5–50 ng) of dsDNA template
*X μL (125 ng) of each oligonucleotide primer
**If primers are greater than 20% different in length, scale the amount of primer added so that primer is added in approximately equimolar amounts. See [http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Multi Site-Directed Mutagenesis manual] for details.
*1 μL of dNTP mix (100mM total dNTP mix with 25 mM each individual dNTP)
*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 Multi 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 with 10-15 bp of matching sequence on each side.
#*Primers should be 25-45bp in length with a melting temp of >=75°C. Stratagene recommends not using primers greater than 45 bp in order to avoid formation of secondary structure. Primers should have comparable melting temperatures.
#*See the [http://www.stratagene.com/manuals/200514.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 >=75°C.
#*Primers should have at least 40% GC content and terminate in one or more C or G bases at the 3' end.
#*PAGE purification of primers may improve mutagenesis efficiency. See [[Designing primers#Oligo synthesis information | here]] for links to information on oligo purification.
#*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).
##68°C for 20 mins
#*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.
#[[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.

==Notes==
*Stratagene does not recommend this protocol for insertions or deletions.
*Apparently there are no primer spacing-dependent effects on mutagenesis efficiency (primers can be adjacent or far apart).

==Reference==
[http://www.stratagene.com/manuals/200514.pdf QuikChange® Multi Site Directed Mutagenesis Kit]

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