PNK Treatment of DNA Ends

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*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.
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*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.
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*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)==

Revision as of 16:59, 24 January 2006

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 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.
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