Team:Alberta/Notebook/protocols/digest

Restriction Digest

Reagents:

  • Restriction enzyme buffer
    • Buffer information can be found at www.NEB.com using the NEBtools.
  • 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
    • Bovine Serum Albumin-BSA (if needed)
    • DNA
    • Enzyme I
    • Enzyme II
  • An example of a typical 25 ul reaction would be:
MilliQ water 15.5 ul
10x NEBuffer 2.5 ul
DNA (200 ng/ul) 5.0 ul
XbaI 1.0 ul
PstI 1.0 ul
  • Incubate at 37oC for one hour (longer is okay too).


Notes:

FastDigest (by Fermentas) enzymes use a single uniform buffer, and claim to work in 5 minutes.


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