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Ligations of plasmid DNA to insert DNA

A typical ligation reaction consists of about 20-200 ng of a vector and a 1-3 fold molar excess of insert DNA. A typical ligation reaction consists of the following:

  • 20-100 ng vector
  • 3-fold MOLAR excess (not mass) of insert DNA
  • 4 μl 5X ligase buffer or 2 μl 10X ligase buffer (note: ligase buffer contains ATP and is unstable when repeatedly frozen and thawed. Prepare small aliquots of buffer and discard aliquot after use)
  • water to 19 μl
  • 1 μl T4 DNA ligase

Incubate at room temperature for 2-24 hours. For transformation by electroporation, ethanol precipitate as described in the transformation procedure.

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Last updated on 3/2/2010

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