Team:Alberta/Project/Digest

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University of Alberta - BioBytes










































































































Restriction Digest

What you will need:

  • Restriction enzyme buffer
  • Restriction enzymes
  • milliQ water
  • DNA to be digested

Procedure:

  • Select a restriction enzyme buffer that is appropriate for BOTH of the enzymes you are using. See information sheets at front of lab for correct buffer and concentration.
  • The total volume of all enzymes in the reaction should be less than 10% of the final reaction volume. Enzymes usually are supplied at 10U/ul and 1ul will be more than enough for our digests.
  • Add components in the following order:
    • Water
    • Buffer
    • DNA
    • Enzyme I
    • Enzyme II
  • An example of a typical 25 ul reaction would be
    • milliQ water 15.5 ul
    • 10x Tango Buffer 2.5 ul
    • DNA (200 ng/ul) 5.0 ul
    • XbaI 1.0 ul
    • PstI 1.0 ul
  • Incubate at 37 C for two hours (longer is okay too).

Notes

  • FastDigest enzymes use a single uniform buffer, and claim to work in 5 min.