Phosphatase treatment of linearized vector - Revision history

Reshma P. Shetty at 12:49, 20 March 2007

Reshma P. Shetty — Tue, 20 Mar 2007 12:49:51 GMT

←Older revision		Revision as of 12:49, 20 March 2007
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	#Incubate 60 mins at 37°C. <br> This should be sufficient to remove 5' phosphates even from 5' recessed ends like those produced by Pst I.		#Incubate 60 mins at 37°C. <br> This should be sufficient to remove 5' phosphates even from 5' recessed ends like those produced by Pst I.
	#Heat-inactivate for 5 mins at 65°C.		#Heat-inactivate for 5 mins at 65°C.
-	#Proceed directly to [[~~Ligation~~ \| ligation]] step.	+	#Proceed directly to [[DNA ligation \| ligation]] step.

	==Notes==		==Notes==

Jenny T Nguyen at 18:52, 10 July 2006

Jenny T Nguyen — Mon, 10 Jul 2006 18:52:14 GMT

←Older revision		Revision as of 18:52, 10 July 2006
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	[http://www.neb.com/nebecomm/products/productM0289.asp NEB's Antarctic Phosphatase]		[http://www.neb.com/nebecomm/products/productM0289.asp NEB's Antarctic Phosphatase]
		+
		+	[[Category:Protocol]]
		+	[[Category:DNA]]
		+	[[Category:In vitro]]

Jason R. Kelly: /* Materials */

Jason R. Kelly — Thu, 25 Aug 2005 21:04:25 GMT

Materials

←Older revision		Revision as of 21:04, 25 August 2005
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	==Materials==		==Materials==

-	*Linear DNA from [[Restriction ~~Digest~~ \| restriction digest]] (heat-inactivation of restriction enzymes is necessary but [[Purification of DNA \| DNA purification]] is not).	+	*Linear DNA from [[Restriction digest \| restriction digest]] (heat-inactivation of restriction enzymes is necessary but [[Purification of DNA \| DNA purification]] is not).
	*[[Antarctic Phosphatase]]		*[[Antarctic Phosphatase]]
	*10X Antarctic Phosphatase buffer		*10X Antarctic Phosphatase buffer

Reshma P. Shetty: /* Materials */

Reshma P. Shetty — Wed, 01 Jun 2005 14:13:16 GMT

Materials

←Older revision		Revision as of 14:13, 1 June 2005
Line 3:		Line 3:
	==Materials==		==Materials==

-	*Linear DNA from [[Restriction Digest]] (heat-inactivation of restriction enzymes is necessary but [[Purification of DNA \| DNA purification]] is not).	+	*Linear DNA from [[Restriction Digest \| restriction digest]] (heat-inactivation of restriction enzymes is necessary but [[Purification of DNA \| DNA purification]] is not).
	*[[Antarctic Phosphatase]]		*[[Antarctic Phosphatase]]
	*10X Antarctic Phosphatase buffer		*10X Antarctic Phosphatase buffer

Reshma P. Shetty at 16:52, 31 May 2005

Reshma P. Shetty — Tue, 31 May 2005 16:52:04 GMT

New page

To minimize self-ligated vector in your transformation, treat your linearized vector with a phosphatase to remove the 5' phosphates necessary for ligation. This should improve the percentage of colonies with inserts.

==Materials==

*Linear DNA from [[Restriction Digest]] (heat-inactivation of restriction enzymes is necessary but [[Purification of DNA | DNA purification]] is not).
*[[Antarctic Phosphatase]]
*10X Antarctic Phosphatase buffer

==Procedure==

#Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample.
#Add 1μL Antarctic Phosphatase (probably should make final glycerol concentration less that 5%?)
#Incubate 60 mins at 37°C. <br> This should be sufficient to remove 5' phosphates even from 5' recessed ends like those produced by Pst I.
#Heat-inactivate for 5 mins at 65°C.
#Proceed directly to [[Ligation | ligation]] step.

==Notes==

[http://www.neb.com/nebecomm/products/productM0289.asp NEB's Antarctic Phosphatase]