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Revision as of 13:08, 29 June 2012 by Sean P Corum (talk | contribs) (Procedure)


Standard DNA single or double digest of variable volume V using NEB restriction enzymes.


  • X μL DNA template
  • NEB Digestion Buffer # (# = 1, 2, 3, or 4)
  • 10mg/ml BSA


  1. Determine compatibility and reaction conditions using Double Digest Finder (double digest only).
  2. Determine reaction volume V by computing V = [(X + 1)/0.8] μL (single digest) or V = [(X + 2)/0.8] μL (double digest).
  3. To DNA sample, add
    • 0.1V 10X Buffer Y
    • 0.1V 10mg/ml BSA
    • 1 μL restriction enzyme 1
    • 1 μL restriction enzyme 2 (double digest only)
  4. Incubate 3 hr to overnight at reaction temperature given by NEB (standard = 37 °C).
  5. Optional: Heat inactivate restriction enzyme(s) by incubation 80 °C 20 min. Not necessary when digestion product is going to be purified or gel extracted, as these procedures remove the enzymes.


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.

Digital Signature

  • SC 14:15, 29 June 2012 (EDT):