Restriction Enzyme Digestion of Plasmid DNA

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Restriction Enzyme Digestions of Plasmid DNA

Restriction Endonuclease Digestions - volumes to use:
DNA 2uL (sufficient for checking minipreps)
10x buffer 1uL (use 1.5uL for 15 uL digestions, and so on)
Enzyme 1uL
water up to 10uL

digest for at least 1 hour; if quantitative digestion is required (e.g. digesting PCR products for cloning), digest overnight

· when pipetting out the enzyme, always keep it in the freezing block, or else it’ll easily lose activity
· also be careful to try to remove residual glycerol on the pipet tip, as more than 10% glycerol may decrease the efficiency of the enzymatic reaction

Check the New England Biolabs catalogue to determine
if BSA is required
which 10x reaction buffer to use
some digestions may require incubation in temperatures other than 37 degrees
how long to digest restriction enzyme sites that are located near the end of a linear fragment

a stock solution of 10x BSA may be made by diluting the 100x BSA provided by New England Biolabs to 10x BSA

a master mix may be prepared by mixing in everything except the DNA, and then aliquoting out the master mix for each individual DNA reaction

once the reaction is completed, add 2uL of 6x loading dye, and run on a gel

for blunt end-ligations, it is necessary to check the orientation of the insert with a different enzyme

if subsequently performing a ligation, it may be prudent to add alkaline phosphatase to the sample (1h, 37 degrees, in either NEBuffer 2, 3, or 4)

Related page of interest: Books with Molecular Biology Protocols