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

Felix Moser: /* Procedure */

2009-08-05T20:41:52Z

Procedure

←Older revision		Revision as of 20:41, 5 August 2009
Line 22:		Line 22:
	##94°C for 5 mins		##94°C for 5 mins
	##94°C for 30 seconds		##94°C for 30 seconds
-	##56°C for 30 seconds (or whatever an appropriate annealing temperature is)	+	##55°C for 30 seconds (or whatever an appropriate annealing temperature is)
-	##68°C for 30 seconds	+	##72°C for 30 seconds
	##Repeat steps 2-4 2-3 cycles		##Repeat steps 2-4 2-3 cycles
-	##68°C for 5 mins	+	##72°C for 5 mins
	#Use fresh 1μL PCR product in a [[Knight:TOPO TA cloning\|TOPO TA cloning reaction]].		#Use fresh 1μL PCR product in a [[Knight:TOPO TA cloning\|TOPO TA cloning reaction]].

Reshma P. Shetty at 22:59, 9 May 2007

2007-05-09T22:59:52Z

←Older revision		Revision as of 22:59, 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: /* Notes */

2006-10-19T23:49:58Z

Notes

←Older revision		Revision as of 23:49, 19 October 2006
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	*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.		*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.
	*'''[[User:Rshetty\|RS]] 21:30, 14 June 2006 (EDT)''': I recently have been trying to make a promoter via this method. I sequenced 3 different clones and all of them had a 1bp deletion in the same position. This was despite the fact that I had ordered my primers to be PAGE purified. I emailed Invitrogen and they said that they will resynthesize and ship me a new primer free of charge. We'll see what happens.		*'''[[User:Rshetty\|RS]] 21:30, 14 June 2006 (EDT)''': I recently have been trying to make a promoter via this method. I sequenced 3 different clones and all of them had a 1bp deletion in the same position. This was despite the fact that I had ordered my primers to be PAGE purified. I emailed Invitrogen and they said that they will resynthesize and ship me a new primer free of charge. We'll see what happens.
-		+	*'''[[User:Rshetty\|Reshma]] 19:49, 19 October 2006 (EDT)''': Tom 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==
	<biblio>		<biblio>
	#Stemmer-Gene-1995 pmid=7590320		#Stemmer-Gene-1995 pmid=7590320
	</biblio>		</biblio>

Reshma P. Shetty at 01:30, 15 June 2006

2006-06-15T01:30:20Z

←Older revision		Revision as of 01:30, 15 June 2006
Line 30:		Line 30:
	==Notes==		==Notes==
	*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.		*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.
-		+	*'''[[User:Rshetty\|RS]] 21:30, 14 June 2006 (EDT)''': I recently have been trying to make a promoter via this method. I sequenced 3 different clones and all of them had a 1bp deletion in the same position. This was despite the fact that I had ordered my primers to be PAGE purified. I emailed Invitrogen and they said that they will resynthesize and ship me a new primer free of charge. We'll see what happens.
		+
	==References==		==References==
	<biblio>		<biblio>
	#Stemmer-Gene-1995 pmid=7590320		#Stemmer-Gene-1995 pmid=7590320
	</biblio>		</biblio>

Reshma P. Shetty at 19:07, 26 May 2006

2006-05-26T19:07:52Z

←Older revision		Revision as of 19:07, 26 May 2006
Line 3:		Line 3:
	==Materials==		==Materials==

-	*Two oligos which overlap by ~20 bp. See [[Knight:Annealing and primer extension with Taq polymerase#Notes\|notes]] for more information on primer ordering. See [[Knight:TOPO TA cloning#Notes\|notes]] for more information on efficient addition of 3'A to PCR products.	+	*Two oligos which overlap by ~20 bp.

	Oligo 1:    5' ----------------------------------- 3'<br>		Oligo 1:    5' ----------------------------------- 3'<br>
Line 11:		Line 11:

	==Procedure==		==Procedure==
-	#Dilute the two oligos to a concentration of 25 μM using H<sub>2</sub>O	+	#Dilute the two oligos to a concentration of 25 μM using H<sub>2</sub>O.
		+	#*General information on [[Designing primers\|primer design]].
		+	#*Notes on [[Knight:Annealing and primer extension with Taq polymerase#Notes\|ordering long primers]].
		+	#*Notes on [[Knight:TOPO TA cloning#Notes\|efficient addition of 3'A to PCR products]].
	#Mix the following in a 0.6 mL sterile tube		#Mix the following in a 0.6 mL sterile tube
	#*9 μL PCR supermix		#*9 μL PCR supermix

Reshma P. Shetty at 18:50, 26 May 2006

2006-05-26T18:50:09Z

New page

This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp) using Taq polymerase. The DNA fragment can be immediately used in a TA cloning reaction. (To proceed to a restriction digest step, [[Purification of DNA|purification]] is necessary.)

==Materials==

*Two oligos which overlap by ~20 bp. See [[Knight:Annealing and primer extension with Taq polymerase#Notes|notes]] for more information on primer ordering. See [[Knight:TOPO TA cloning#Notes|notes]] for more information on efficient addition of 3'A to PCR products.

Oligo 1:    5' ----------------------------------- 3'<br>
Oligo 2:                                        3' ----------------------------------- 5'

*[http://www.invitrogen.com/content/sfs/manuals/11306016.pdf PCR supermix]

==Procedure==
#Dilute the two oligos to a concentration of 25 μM using H<sub>2</sub>O
#Mix the following in a 0.6 mL sterile tube
#*9 μL PCR supermix
#*0.5 μL oligo 1
#*0.5 μL oligo 2
#Anneal and extend the two oligos together by placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) and running the following protocol.
##94°C for 5 mins
##94°C for 30 seconds
##56°C for 30 seconds (or whatever an appropriate annealing temperature is)
##68°C for 30 seconds
##Repeat steps 2-4 2-3 cycles
##68°C for 5 mins
#Use fresh 1μL PCR product in a [[Knight:TOPO TA cloning|TOPO TA cloning reaction]].

==Notes==
*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.

==References==
<biblio>
#Stemmer-Gene-1995 pmid=7590320
</biblio>