• 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.

Prepare reaction mix

1. 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.
2. Add 5 uL of appropriate NEBuffer to each tube.
3. Add 0.5 uL of BSA to each tube.
4. 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.
5. Add 1 uL of the second appropriate restriction enzyme to each tube.
6. 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.

Incubations

1. Incubate the restriction digests at 37 degrees Celsius for at least 1 hour. You can leave this overnight (15 hours).
2. 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.