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 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)

• 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 has 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.