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.