Knight:Annealing and primer extension with Klenow polymerase - Revision history

Reshma P. Shetty at 23:00, 9 May 2007

Reshma P. Shetty — Wed, 09 May 2007 23:00:27 GMT

←Older revision		Revision as of 23:00, 9 May 2007
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	#Stemmer-Gene-1995 pmid=7590320		#Stemmer-Gene-1995 pmid=7590320
	</biblio>		</biblio>
		+
		+	[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]

Reshma P. Shetty: Annealing and primer extension moved to Knight:Annealing and primer extension with Klenow polymerase: Knight lab protocol

Reshma P. Shetty — Thu, 26 Oct 2006 16:28:39 GMT

Annealing and primer extension moved to Knight:Annealing and primer extension with Klenow polymerase: Knight lab protocol

←Older revision

Revision as of 16:28, 26 October 2006

Reshma P. Shetty at 23:50, 19 October 2006

Reshma P. Shetty — Thu, 19 Oct 2006 23:50:36 GMT

←Older revision		Revision as of 23:50, 19 October 2006
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	*The Klenow (exo-) polymerase will exhibit >75% activity at 25°C, compared to 100% at 37°C. Courtesy of Chris Benoit at NEB Technical Support.		*The Klenow (exo-) polymerase will exhibit >75% activity at 25°C, compared to 100% at 37°C. Courtesy of Chris Benoit at NEB Technical Support.
	*If you are annealing two oligos with a region of complimentarity on each end generating an annealed, fully extended product in the 100 bp range, I recommend 5U of Klenow(exo-) per microgram of primed template. Courtesy of Chris Benoit at NEB Technical Support.		*If you are annealing two oligos with a region of complimentarity on each end generating an annealed, fully extended product in the 100 bp range, I recommend 5U of Klenow(exo-) per microgram of primed template. Courtesy of Chris Benoit at NEB Technical Support.
		+	*'''[[User:Rshetty\|Reshma]] 19:50, 19 October 2006 (EDT)''': [[Tom Knight]] suggests considering how much enzyme you use relative to how much primer. Supposedly, polymerase does not necessarily fall off the ends of the DNA which means in 2-3 cycles, very few of your annealed primers will become completely double-stranded (as this requires two polymerases to bind to the same annealed primer molecule and extend). This lack of complete double stranding could increase the potential for errors.

	==References==		==References==

Barry Canton: /* Calculating amount of oligo for reaction */

Barry Canton — Sun, 28 May 2006 17:14:02 GMT

Calculating amount of oligo for reaction

←Older revision		Revision as of 17:14, 28 May 2006
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	==Calculating amount of oligo for reaction==		==Calculating amount of oligo for reaction==
-
-	~~''This should be checked for errors'' -[[User:Rshetty\|Reshma]] 19:03, 12 May 2005 (EDT)~~

	<math> \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock}</math>		<math> \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock}</math>

Reshma P. Shetty at 18:56, 26 May 2006

Reshma P. Shetty — Fri, 26 May 2006 18:56:50 GMT

←Older revision		Revision as of 18:56, 26 May 2006
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	This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp). The DNA fragment is prepared for cloning by restriction digest.		This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp). The DNA fragment is prepared for cloning by restriction digest.
		+
		+	Also see [[Knight:Annealing and primer extension with Taq polymerase]] for generation for short DNA fragments for TA cloning.

	==Materials==		==Materials==

Barry Canton: /* Procedure */

Barry Canton — Thu, 11 May 2006 15:01:56 GMT

Procedure

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	#*Y μL oligo 2 (typically 1 μg or more)		#*Y μL oligo 2 (typically 1 μg or more)
	#*(87 - X - Y) μL deionized sterile H<sub>2</sub>O		#*(87 - X - Y) μL deionized sterile H<sub>2</sub>O
-	#Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 65°C, ~~65°C~~ for 5 mins, then a cool down ~~for~~ 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.	+	#Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 5°C below the melting temperature of the primers, hold that temperature for 5 mins, then cool down at 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.
	#Add 1 μL Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase to mixture. <br> Vortex polymerase before pipetting to ensure it is well-mixed.		#Add 1 μL Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase to mixture. <br> Vortex polymerase before pipetting to ensure it is well-mixed.
	#Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP). <br> ''Recommend using a thermal cycler for the following incubation steps.''		#Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP). <br> ''Recommend using a thermal cycler for the following incubation steps.''

Barry Canton: /* Procedure */

Barry Canton — Wed, 10 May 2006 02:10:55 GMT

Procedure

←Older revision		Revision as of 02:10, 10 May 2006
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	#Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP). <br> ''Recommend using a thermal cycler for the following incubation steps.''		#Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP). <br> ''Recommend using a thermal cycler for the following incubation steps.''
	#Incubate 1 hr at 37°C.		#Incubate 1 hr at 37°C.
-	#Heat inactivate polymerase by incubating at 75°C for 20 minutes. <br> ''See [[Restriction Digest]] for more information on the following steps.''	+	#Heat inactivate polymerase by incubating at 75°C for 20 minutes. This inactivation temperature might be higher than the melting temperature of your annealed and extended primers. It may be prudent to ramp the temperature down from 75°C. <br> ''See [[Restriction Digest]] for more information on the following steps.''
	#Add 1 μL restriction enzyme(s) to mixture.		#Add 1 μL restriction enzyme(s) to mixture.
	#Incubate for a minimum of 2 hrs.		#Incubate for a minimum of 2 hrs.

Reshma P. Shetty: /* References */

Reshma P. Shetty — Thu, 04 May 2006 17:27:20 GMT

References

←Older revision		Revision as of 17:27, 4 May 2006
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	==References==		==References==
-		+	<biblio>
-	~~W. P.~~ Stemmer~~, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single~~-~~step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides.~~ Gene~~, 164(1):49–53,~~ 1995~~. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids~~=7590320~~&dopt=Abstract PubMed]~~	+	#Stemmer-Gene-1995 pmid=7590320
		+	</biblio>

Reshma P. Shetty: /* Notes */

Reshma P. Shetty — Thu, 04 May 2006 17:26:21 GMT

Notes

←Older revision		Revision as of 17:26, 4 May 2006
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	*Klenow is tolerant of a broad range buffer conditions. This includes NEBuffer for EcoRI in which it will exhibit a rate of polymerization of approximately equal to 50% of that in its recommended buffer. For greater reproducibilty, you could add DTT to the reaction, since DTT is absent from EcoRI reaction buffer. Courtesy of Paul Walsh at NEB Technical Support.		*Klenow is tolerant of a broad range buffer conditions. This includes NEBuffer for EcoRI in which it will exhibit a rate of polymerization of approximately equal to 50% of that in its recommended buffer. For greater reproducibilty, you could add DTT to the reaction, since DTT is absent from EcoRI reaction buffer. Courtesy of Paul Walsh at NEB Technical Support.
	*When using Klenow(exo-) for this type of reaction, there is much less risk of "overdoing" the reaction than when using regular Klenow. Incubating at 37C for one hour, using one unit of Klenow(exo-) per pmol of DNA should result in the desire 90bp duplex. Courtesy of Paul Walsh at NEB Technical Support.		*When using Klenow(exo-) for this type of reaction, there is much less risk of "overdoing" the reaction than when using regular Klenow. Incubating at 37C for one hour, using one unit of Klenow(exo-) per pmol of DNA should result in the desire 90bp duplex. Courtesy of Paul Walsh at NEB Technical Support.
		+	*The Klenow (exo-) polymerase will exhibit >75% activity at 25°C, compared to 100% at 37°C. Courtesy of Chris Benoit at NEB Technical Support.
		+	*If you are annealing two oligos with a region of complimentarity on each end generating an annealed, fully extended product in the 100 bp range, I recommend 5U of Klenow(exo-) per microgram of primed template. Courtesy of Chris Benoit at NEB Technical Support.

	==References==		==References==

	W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49–53, 1995. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=7590320&dopt=Abstract PubMed]		W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49–53, 1995. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=7590320&dopt=Abstract PubMed]

Barry Canton: /* Notes */

Barry Canton — Tue, 07 Mar 2006 22:09:32 GMT

Notes

←Older revision		Revision as of 22:09, 7 March 2006
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	==Notes==		==Notes==

-	*~~Once~~ oligos ~~become about around~~ 50-60 bp in length, there can often be problems with errors or deletions in the primers. Therefore, it might be worth ordering your primers with an extra purification step such as PAGE. [https://catalog.invitrogen.com/index.cfm?fuseaction=customerSite.primersHome Invitrogen custom primers] offers this service for an extra fee.	+	*For oligos greater than 50-60 bp in length, there can often be problems with errors or deletions in the primers. Therefore, it might be worth ordering your primers with an extra purification step such as PAGE. [https://catalog.invitrogen.com/index.cfm?fuseaction=customerSite.primersHome Invitrogen custom primers] offers this service for an extra fee.
	*Klenow is tolerant of a broad range buffer conditions. This includes NEBuffer for EcoRI in which it will exhibit a rate of polymerization of approximately equal to 50% of that in its recommended buffer. For greater reproducibilty, you could add DTT to the reaction, since DTT is absent from EcoRI reaction buffer. Courtesy of Paul Walsh at NEB Technical Support.		*Klenow is tolerant of a broad range buffer conditions. This includes NEBuffer for EcoRI in which it will exhibit a rate of polymerization of approximately equal to 50% of that in its recommended buffer. For greater reproducibilty, you could add DTT to the reaction, since DTT is absent from EcoRI reaction buffer. Courtesy of Paul Walsh at NEB Technical Support.
	*When using Klenow(exo-) for this type of reaction, there is much less risk of "overdoing" the reaction than when using regular Klenow. Incubating at 37C for one hour, using one unit of Klenow(exo-) per pmol of DNA should result in the desire 90bp duplex. Courtesy of Paul Walsh at NEB Technical Support.		*When using Klenow(exo-) for this type of reaction, there is much less risk of "overdoing" the reaction than when using regular Klenow. Incubating at 37C for one hour, using one unit of Klenow(exo-) per pmol of DNA should result in the desire 90bp duplex. Courtesy of Paul Walsh at NEB Technical Support.