From 2007.igem.org

Vector digestion

Use X units of enzymes per 1ug of DNA (over digesting by factor of X) Usually we use over digest factor of 10 unless otherwise specified in the enzyme tech sheet. If over digestion results in star activity use 3X

Calculate the volume required for each

DNA µg = DNA volume * concentration

Enzyme volume = Enzyme unit/µl* # units = X [µl] Buffer is dilution factor x dilution of the total volume. [i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume] Order of filling

DNA

Water

Buffer

Enzyme

Incubate for 3 hours at the specified temperature for the enzyme and deactivate at the appropriate temperature for 20 mins.

Please keep the buffer on ice at all times when out of -20C. Keep the enzyme in the benchtop coolers at all times when out of the -20C.