Difference between revisions of "PNK Treatment of DNA Ends"

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This step 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 step 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.
  
*T4 Polynucleotide Kinase (available from [http://www.neb.com NEB])is an enzyme that can perform this on blunt or overhanging DNA ends.
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*[http://www.neb.com/nebecomm/products/productM0201.asp T4 Polynucleotide Kinase] is an enzyme that can perform this on blunt or overhanging DNA ends.
  
*They describe a protocol for use which is summarized in a modified form below.
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*They describe a protocol for use that is modified and summarized below.
 +
 
 +
*Reaction Mix
 +
**1<math>\mu</math>l PNK stock (10,000U/ml)
 +
**1<math>\mu</math>l T4 Ligase Buffer
 +
**8<math>\mu</math>l Substrate
 +
 
 +
*Reaction Conditions
 +
*37<sup>o</sup>C for 30mins
 +
*65<sup>o</sup>C for 20mins
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*Store at 4<sup>o</sup>C
 +
 
 +
*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 18:47, 3 May 2005

This step 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.

  • They describe a protocol for use that is modified and summarized below.
  • Reaction Mix
    • 1[math]\mu[/math]l PNK stock (10,000U/ml)
    • 1[math]\mu[/math]l T4 Ligase Buffer
    • 8[math]\mu[/math]l Substrate
  • Reaction Conditions
  • 37oC for 30mins
  • 65oC for 20mins
  • Store at 4oC
  • 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.