Restriction Digest

This protocol was adapted from GinkgoBioworks BioBrick Assembly Manual.

  • nuclease-free water
  • 500 ng of DNA (you must calculate this from the concentration)
  • NEBuffer (for X/P- NEBuffer 3, for E/S, E/P, E/X, S/P- NEBuffer 4)
  • Restriction enzyme 1
  • Restriction enzyme 2
  • Turn on a water bath to 37 degrees Celcius.
  • Turn on a heat block to 80 degrees Celsius.
  • Get a bucket of ice and put the materials in the ice.
  1. Prepare the reaction mix
    • To each tube, add nuclease-free water and 500 ng of the part or plasmid to be /digested. Adjust the amount of water you add such that the total value in each tube is 42.5 uL. We usually add the the water before the DNA.
    • Add 5 uL of appropriate NEBuffer to each tube.
    • Add 0.5 uL of BSA to each tube.
    • Add 1 uL of the first appropriate restriction enzyme to each tube. When pipetting restriction enzyme, only touch the very end of the pipet tip into the restriction enzyme. Restriction enzymes are stored in a high percentage glycerol solution that sticks to the outside of the pipet tip; if you dip the tip deeply into the restriction digest you will add much more restriction digest than needed as well as increase the glycerol concentration of the digest mix.
    • Add 1 uL of the second appropriate restriction enzyme to each tube.
    • The total volume in each tube should now be 50 uL. Ensure the digest is well-mixed by flicking the tube or pipetting up and down. You can spin the tub in a microcentrifuge to collect the liquid at the bottom of the tube.
  2. Incubations
    • Incubate the restriction digests at 37 degrees Celsius for at least 1 hour. You can leave this overnight (15 hours).
    • Incubate the restriction digests at 80 degrees Celsius for 20 min to deactivate the restriction enzymes.
Final steps
  • Run a gel with your digests to make sure you have bands of expected length. This will ensure that the digest worked as expected.
  • Store unused digests at -20 degrees C.