Corum:Digestion

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Revision as of 12:46, 26 July 2012 by Sean P Corum (Talk | contribs)
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Contents

Overview

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

Materials

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

Procedure

  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 NEB Buffer Y (Y = 1, 2, 3, or 4)
    • 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.


  • Lab standard double digestion uses 22 μL plasmid DNA, so V = (22 + 2)/0.8 μL = 30 μL.
    • 22 μL plasmid DNA
    • 3 μL 10X NEB Buffer Y
    • 3 μL 10mg/ml BSA
    • 1 μL restriction enzyme 1
    • 1 μL restriction enzyme 2

Notes

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

  1. List troubleshooting tips here.
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  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers, books, and websites

  1. New England Biolabs

Contact

or instead, discuss this protocol.

Digital Signature

  • SC 14:15, 29 June 2012 (EDT):
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