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.