(Difference between revisions)
Line 47: Line 47:
|PstI      ||    1.0 ul
|PstI      ||    1.0 ul
*Incubate at 37<sup>o</sup>C for one hours (longer is okay too).
*Incubate at 37<sup>o</sup>C for one hour (longer is okay too).

Revision as of 04:24, 26 October 2010


Protocol 5: Restriction Digest


  • Restriction enzyme buffer
    • Buffer information can be found at using the NEBtools.
  • Restriction enzymes
  • MilliQ water
  • DNA to be digested


  • 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).


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