PNK Treatment of DNA Ends

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__NOTOC__
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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.
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.
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*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.
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*10 <math>\mu</math>L Reaction Mix
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==Reaction Mix==
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*10 <math>\mu</math>L Mix
**1 &mu;L PNK stock (10,000 U/ml)
**1 &mu;L PNK stock (10,000 U/ml)
**1 &mu;L T4 Ligase Buffer
**1 &mu;L T4 Ligase Buffer
**8 &mu;L Substrate
**8 &mu;L Substrate
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*Reaction Conditions
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==Reaction Conditions==
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*37&deg;C for 30mins
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#37&deg;C for 30mins
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*65&deg;C for 20mins
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#65&deg;C for 20mins
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*Store at 4&deg;C
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#Store at 4&deg;C
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==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.
*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.

Revision as of 17:01, 29 September 2005

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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