Team:Alberta/Notebook/protocols/digest

From 2010.igem.org

TEAM ALBERTA

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:

• Incubate at 37^oC for one hour (longer is okay too).

Notes:

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