PNK Treatment of DNA Ends

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Revision as of 17:01, 29 September 2005 by Barry Canton (Talk | contribs)
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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.

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

Reaction Mix

  • 10 μL Mix
    • 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 the author has not done this.
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