Restriction Enzyme Digestion of Plasmid DNA

Restriction endonuclease Digestions – volume of use:

DNA 2uL (enough to check minipreps)
10x buffer 1UL (use 1.5uL to 15 digestions uL, and so on)
enzyme 1UL
water to 10ul

digest for at least 1 hour; if the quantitative digestion is required (eg digested PCR product for cloning), digest overnight.

Restriction Enzyme Digestion of Plasmid DNA

when Pipetting out the enzyme, always remains in the block freezing, or else itll easily lose their activity
Also be careful to try to remove the residual glycerol in the pipette tip, because more than 10% of glycerol can reduce the efficiency of enzymatic reactions

Check the New England Biolabs catalog to determine
if the BSA is required
the 10x reaction buffer for use
some digestions may require incubation in a temperature other than 37 degrees
how long to digest the restriction enzyme sites located near the ends of the linear fragment

BSA 10x stock solution can be made by dilution 100x BSA supplied by New England Biolabs 10x BSA

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

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

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

if then perform ligation, it may be wise to add to samples of alkaline phosphatase (1h, 37 degrees, both NEBuffer 2, 3, or 4)