Most researchers follow the general rule that 10 units of restriction endonuclease is sufficient to overcome variability in DNA source, quantity and purity. Generally, 1 μl of enzyme is added to 1 μg of purified DNA in a final volume of 50 μl of the appropriate 1X NEBuffer followed by incubation for 1 hour at the recommended temperature. If an excess of enzyme is used, the length of incubation can often be decreased to save time. Alternatively, you can productively digest with fewer units of enzyme for up to 16 hours with many restriction enzymes.
To keep glycerol concentration at less than 5% in a reaction, the restriction enzyme, which is supplied in 50% glycerol, should not exceed 10% of the total reaction volume
An extremely important, yet often overlooked, element of a successful restriction digest is mixing. The reaction must be thoroughly mixed to achieve complete digestion. We recommend gently pipetting ther reaction mixture up and down or "flicking" the reaction tube. Follow with a quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.