PNK Treatment of DNA Ends

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 (10&mu;l)

 * 1 &mu;L PNK stock (10,000 U/ml)
 * 1 &mu;L T4 Ligase Buffer
 * 8 &mu;L Substrate

Reaction Conditions

 * 1) 37&deg;C for 30mins
 * 2) 65&deg;C for 20mins
 * 3) Store at 4&deg;C

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

Text Output
PNK Treatment of DNA Ends protocol

Source Code
PNK Treatment of DNA Ends protocol - source code