The restriction enzyme digest is a common molecular biology technique used to cut DNA at specific sequences. This will result in fragments of an expected size, which can then be used to validate genetic products or to produce DNA fragments for further use. What follows is a standard protocol (adapted from NEB) for a restriction enzyme digest of a plasmid which can be modified by the end user in a myriad of ways.

Materials

  1. Product which requires digest (i.e. plasmid with insert of interest)
  2. Heat block, water bath, or incubator which can maintain a temperature of 37°C.
  3. Enzymes
  4. Sterilized and filtered water
  5. EDTA

Protocol

  1. Obtain concentration of plasmid. Identify restriction enzyme sites. Estimate size of final products.
    1. Snapgene Viewer is perfect for this task.
  2. Prepare the following into a clean microcentrifuge tube. The final reaction volume is 20uL but reaction volumes can range from 10 to 50uL depending on the situation. Just multiply volumes as necessary.
Reagent Volume/Mass Notes
DNA 1ug Can also use 0.5ug for diagnostic digests
10x rCutSmart 2uL Different buffers can be used (rarer nowadays), rCutSmart works for almost all reactions, however
Enzyme 1 1uL
Enzyme 2 1uL
Sterilized MilliQ Volume up to 20uL Filtered MilliQ also works
  1. Spin down contents in centrifuge for 1 min at 14K rpm.
  2. Transfer contents to heat block at 37°C
  3. Heat for 1 hour
    1. Time can be variable. For quick, diagnostic digests 15 minutes is satisfactory. For difficult digests with substantial amounts of uncut plasmid (supercoiled), very long products, or nicked products the digest time can be increased by several hours. Overnight digests can be done, but beware of star activity (relaxation of cutting fidelity) that may occur
  4. Add 0.5uL of 0.5M EDTA pH 8.0. Spin down sample.
    1. Most loading buffers contain EDTA, but I always add a small amount to quench the digest.
  5. Digest can be frozen at this point.

Additional Notes