Rao:Restriction Digest

Overview

Cleavage of DNA by restriction endonucleases is a crucial component of any aspect of biological research. From genetics to protein analysis it is something you cannot avoid. Here's a generally accepted protocol in the Rao Lab.

Material

• Some kind of tube to hold the reaction: 0.8 mL or 1.8 mL microcentrifuge tubes should be fine
• Desired restriction enzymes (we use NEB restriction enzymes)

Method

In your tube add the following:

• x uL DNA solution
• 5 uL 10X Reaction buffer
• 0.5 uL 100X BSA
• x uL H2O
• 1 uL of restriction enzyme(s)

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50 uL total volume

Digest for 1+ hr at 37C (or other temperature specified for your enzyme). Water baths are nice for this, but your incubator is fine too.

Considerations

• BSA is not necessary for all reactions, but it does not appear to hurt anything. In fact some say it is good to use it, especially in double digests (see below).
• Often, when cloning, more than one restriction enzyme are needed be used. While most enzymes are reasonably compatible, some are not. It is best to consult a heuristic based chart like the one in the NEB catalogue.
• When setting up the reaction, make sure addition of addition of enzymes does not get the glycerol concentration in the reaction mixture to over 5%. High glycerol concentrations inhibit endonuclease activities.