This protocol was adapted from GinkgoBioworks BioBrick Assembly Manual.

Materials

• 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

Prepare

• 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.

Procedure

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.