PNK Treatment of DNA Ends - Revision history

Vaishnavi Ananth: /* BioStream version */

2009-11-19T10:01:53Z

BioStream version

←Older revision		Revision as of 10:01, 19 November 2009
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	*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.		*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.

-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the PNK Treatment of DNA Ends protocol in ~~BioStream~~, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in ~~BioStream~~ (see Source code). More information about ~~BioStream~~ can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]	+	Following is the PNK Treatment of DNA Ends protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
	====Text Output====		====Text Output====
	[[PNK Treatment of DNA Ends protocol]]		[[PNK Treatment of DNA Ends protocol]]

Vaishnavi Ananth at 10:04, 1 October 2009

2009-10-01T10:04:02Z

←Older revision		Revision as of 10:04, 1 October 2009
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	*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.		*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.
		+
		+	==BioStream version==
		+	Following is the PNK Treatment of DNA Ends protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
		+	====Text Output====
		+	[[PNK Treatment of DNA Ends protocol]]
		+	====Source Code====
		+	[[PNK Treatment of DNA Ends protocol - source code]]

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

Jason R. Kelly: /* Notes */

2007-12-24T14:39:40Z

Notes

←Older revision		Revision as of 14:39, 24 December 2007
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	==Notes==		==Notes==
-	*The T4 Ligase Buffer provides the required ATP and substitutes for the PNK Buffer and ATP in the NEB protocol. It is actually possible to perform the PNK step and a ligation step simultaneously although [[Barry Canton \| I]] ~~has~~ not done this.	+	*The T4 Ligase Buffer provides the required ATP and substitutes for the PNK Buffer and ATP in the NEB protocol. It is actually possible to perform the PNK step and a ligation step simultaneously although [[Barry Canton \| I]] have not done this.

	*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.		*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.

Jenny T Nguyen at 18:52, 10 July 2006

2006-07-10T18:52:37Z

←Older revision		Revision as of 18:52, 10 July 2006
Line 25:		Line 25:

	*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.		*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.
		+
		+	[[Category:Protocol]]
		+	[[Category:DNA]]
		+	[[Category:In vitro]]

Kathmc at 20:59, 24 January 2006

2006-01-24T20:59:38Z

←Older revision		Revision as of 20:59, 24 January 2006
Line 9:		Line 9:
	*If you plan on PNK treating complementary oligos it is best to do so prior to annealing the oligos since phosphorylation of single-stranded DNA is more efficient (see above) and also because the heat inactivation step may be close to the melting temperature of the annealed oligos.		*If you plan on PNK treating complementary oligos it is best to do so prior to annealing the oligos since phosphorylation of single-stranded DNA is more efficient (see above) and also because the heat inactivation step may be close to the melting temperature of the annealed oligos.

-	*T4 PNK can also be used to phosphorylate RNA, and is commonly used for radiolabeling RNA. Ensure that the enzyme you are using for labeling RNA is RNase-free (this is the case for most commercial enzymes.	+	*T4 PNK can also be used to phosphorylate RNA, and is commonly used for radiolabeling RNA. Ensure that the enzyme you are using for labeling RNA is RNase-free (this is the case for most commercial enzymes).

	==Reaction Mix (10μl)==		==Reaction Mix (10μl)==

Barry Canton at 20:40, 24 January 2006

2006-01-24T20:40:26Z

←Older revision		Revision as of 20:40, 24 January 2006
Line 7:		Line 7:
	*The above website outlines a protocol for use that is modified and summarized below.		*The above website outlines a protocol for use that is modified and summarized below.

-	*If you ~~have annealed primers, make sure to check their melting temperature as~~ it ~~may not be significantly above the heat inactivation step of this protocol. If this~~ is ~~the case, either don't bother with the PNK step, or make sure~~ to ~~cool the reaction mix very slowly after the heat inactivation to allow reannealing of any melted DNA. If possible, the PNK step should be performed~~ prior to annealing of ~~your oligo cassette, as phosphorylation will be more efficient with on the~~ single-stranded DNA (see above).	+	*If you plan on PNK treating complementary oligos it is best to do so prior to annealing the oligos since phosphorylation of single-stranded DNA is more efficient (see above) and also because the heat inactivation step may be close to the melting temperature of the annealed oligos.

	*T4 PNK can also be used to phosphorylate RNA, and is commonly used for radiolabeling RNA. Ensure that the enzyme you are using for labeling RNA is RNase-free (this is the case for most commercial enzymes.		*T4 PNK can also be used to phosphorylate RNA, and is commonly used for radiolabeling RNA. Ensure that the enzyme you are using for labeling RNA is RNase-free (this is the case for most commercial enzymes.

Kathmc: /* Introduction */

2006-01-24T20:30:46Z

Introduction

←Older revision		Revision as of 20:30, 24 January 2006
Line 3:		Line 3:
	This protocol is used to add a phosphate group to the 5' end of a single or double stranded DNA molecule. Most primers, for example, are ordered without this being added as it requires an extra synthesis step and hence greater cost. However subsequent ligation steps are more efficient if these phosphate groups are added.		This protocol is used to add a phosphate group to the 5' end of a single or double stranded DNA molecule. Most primers, for example, are ordered without this being added as it requires an extra synthesis step and hence greater cost. However subsequent ligation steps are more efficient if these phosphate groups are added.

-	*[http://www.neb.com/nebecomm/products/productM0201.asp T4 Polynucleotide Kinase] is an enzyme that can perform this on blunt or overhanging DNA ends.	+	*[http://www.neb.com/nebecomm/products/productM0201.asp T4 Polynucleotide Kinase] is an enzyme that can perform this on blunt or overhanging DNA ends. T4 polynucleotide kinase phosphorylates single-stranded DNA most efficiently, followed by overhanging ends, and then by blunt-ended double-stranded DNA.

	*The above website outlines a protocol for use that is modified and summarized below.		*The above website outlines a protocol for use that is modified and summarized below.

-	*If you have annealed primers, make sure to check their melting temperature as it may not be significantly above the heat inactivation step of this protocol. If this is the case, either don't bother with the PNK step, or make sure to cool the reaction mix very slowly after the heat inactivation to allow reannealing of any melted DNA.	+	*If you have annealed primers, make sure to check their melting temperature as it may not be significantly above the heat inactivation step of this protocol. If this is the case, either don't bother with the PNK step, or make sure to cool the reaction mix very slowly after the heat inactivation to allow reannealing of any melted DNA. If possible, the PNK step should be performed prior to annealing of your oligo cassette, as phosphorylation will be more efficient with on the single-stranded DNA (see above).
		+
		+	*T4 PNK can also be used to phosphorylate RNA, and is commonly used for radiolabeling RNA. Ensure that the enzyme you are using for labeling RNA is RNase-free (this is the case for most commercial enzymes.

	==Reaction Mix (10μl)==		==Reaction Mix (10μl)==

Barry Canton: /* Notes */

2006-01-24T19:02:54Z

Notes

←Older revision		Revision as of 19:02, 24 January 2006
Line 20:		Line 20:

	==Notes==		==Notes==
-	*The T4 Ligase Buffer provides the required ATP and substitutes for the PNK Buffer and ATP in the NEB protocol. It is actually possible to perform the PNK step and a ligation step simultaneously although ~~the author~~ has not done this.	+	*The T4 Ligase Buffer provides the required ATP and substitutes for the PNK Buffer and ATP in the NEB protocol. It is actually possible to perform the PNK step and a ligation step simultaneously although [[Barry Canton \| I]] has not done this.
		+
		+	*Performing this protocol on an insert along with a [[Phosphatase treatment of linearized vector\|phosphatase step]] on the vector can greatly improve the efficiency of a ligation by reducing the likelihood of a vector religating at the same time as making the ligation of vector and insert more likely.

Barry Canton at 18:59, 24 January 2006

2006-01-24T18:59:10Z

←Older revision		Revision as of 18:59, 24 January 2006
Line 1:		Line 1:
	__NOTOC__		__NOTOC__
	==Introduction==		==Introduction==
-	This protocol is used to add a phosphate group to the 5' end of a DNA molecule. Most primers for example are ordered without this being added as it requires an extra synthesis step and hence greater cost. However subsequent ligation steps are more efficient if these phosphate groups are added.	+	This protocol is used to add a phosphate group to the 5' end of a single or double stranded DNA molecule. Most primers, for example, are ordered without this being added as it requires an extra synthesis step and hence greater cost. However subsequent ligation steps are more efficient if these phosphate groups are added.

	*[http://www.neb.com/nebecomm/products/productM0201.asp T4 Polynucleotide Kinase] is an enzyme that can perform this on blunt or overhanging DNA ends.		*[http://www.neb.com/nebecomm/products/productM0201.asp T4 Polynucleotide Kinase] is an enzyme that can perform this on blunt or overhanging DNA ends.

Barry Canton: /* Introduction */

2005-12-09T22:02:40Z

Introduction

←Older revision		Revision as of 22:02, 9 December 2005
Line 6:		Line 6:

	*The above website outlines a protocol for use that is modified and summarized below.		*The above website outlines a protocol for use that is modified and summarized below.
		+
		+	*If you have annealed primers, make sure to check their melting temperature as it may not be significantly above the heat inactivation step of this protocol. If this is the case, either don't bother with the PNK step, or make sure to cool the reaction mix very slowly after the heat inactivation to allow reannealing of any melted DNA.

	==Reaction Mix (10μl)==		==Reaction Mix (10μl)==