Corum:DNA Digestion

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SC 14:53, 19 June 2012 (EDT):


Standard DNA digest protocol using NEB restriction enzymes.


For a DNA digestion of total volume V (in μL):

  • ~0.8V DNA
  • 0.1V 10X digestion buffer (1, 2, 3, or 4; NEB)
  • 0.1V 10mg/ml BSA
  • 1 μL restriction enzyme 1
  • 1 μL restriction enzyme 2 (optional, for double digestion)


  1. Find appropriate enzymes(s), associated buffer, reaction temperature from NEB.
  2. Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
  3. Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
  4. Add the reaction components together in order listed above. Mix gently and spin.
  5. Incubate at reaction temperature for 3 hr to overnight.


Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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Relevant papers, books, and websites

  1. New England Biolabs


or instead, discuss this protocol.