# PNK Treatment of DNA Ends

(Difference between revisions)
 Revision as of 21:51, 3 May 2005 (view source)← Previous diff Current revision (06:01, 19 November 2009) (view source) (→BioStream version) (13 intermediate revisions not shown.) Line 1: Line 1: - 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. + __NOTOC__ + ==Introduction== + 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. - *10$\mu$l Reaction Mix + *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. - **1$\mu$l PNK stock (10,000U/ml) + - **1$\mu$l T4 Ligase Buffer + - **8$\mu$l Substrate + - *Reaction Conditions + *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). - *37oC for 30mins + - *65oC for 20mins + - *Store at 4oC + - *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. + ==Reaction Mix (10μl)== + *1 μL PNK stock (10,000 U/ml) + *1 μL T4 Ligase Buffer + *8 μL Substrate + + ==Reaction Conditions== + #37°C for 30mins + #65°C for 20mins + #Store at 4°C + + ==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]] 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. + + ==BioCoder version== + 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==== + [[PNK Treatment of DNA Ends protocol]] + ====Source Code==== + [[PNK Treatment of DNA Ends protocol - source code]] + + [[Category:Protocol]] + [[Category:DNA]] + [[Category:In vitro]]

## Introduction

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.

• 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.
• 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).

## Reaction Mix (10μl)

• 1 μL PNK stock (10,000 U/ml)
• 1 μL T4 Ligase Buffer
• 8 μL Substrate

## Reaction Conditions

1. 37°C for 30mins
2. 65°C for 20mins
3. Store at 4°C

## 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 I have not done this.
• Performing this protocol on an insert along with a 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.

## BioCoder version

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.Vaishnavi