1. Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
   1. [http://tools.neb.com/NEBcutter2/ NEBcutter] for finding cutting enzymes.
   2. [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double Digest Finder] for the parameters.
2. Calculate the amounts required of:
   1. DNA
   2. Buffer (usually from 10x to 1x)
   3. BSA, if needed (usually from 100x to 1x)
   4. Enzymes (depends on the amount of DNA)
   5. Water
3. Get the recommended buffer (and BSA if needed) from the freezer and let defreeze.
4. Mix all the ingredients, except DNA, in on tube.
5. Note: Enzymes should stay no longer than a couple of minutes out of the freezer. Don't touch the bottom of the tubes! Don't vortex!
6. Distribute the mix in as many tubes as DNA samples and add the DNA.
7. Keep in the Thermomixer at the recommended temperature.

Sowmya's recommended amounts (50 µl total solution):

• 5 µl of 10x buffer
• 0.5 µl of 100x BSA
• 1 µl of each enzyme
• 5 µl of DNA
• 37.5 (up to 50 µl) of water.

Protocol based on what was done on July the 4th.