Principle of restriction digestion
Selecting suitable restriction endonucleases
Compatibility of reaction conditions
Restriction digest components
Setting up a restriction digestion
- Pipet reaction components into a tube and mix well by pipetting.
Tip: Thorough mixing is extremely important.
Tip: The enzyme should be kept on ice and added last.
Tip: When setting up large numbers of digests, make a reaction master mix consisting of water, buffer, and enzyme, and aliquot this into tubes containing the DNA to be digested.
- Centrifuge the tube briefly to collect the liquid at the bottom.
- Incubate the digest in a water bath or heating block, usually for 1–4 h at 37°C. However, some restriction enzymes require higher (e.g., 50–65°C) while others require lower (e.g., 25°C) incubation temperatures.
- For some downstream applications it is necessary to heat-inactivate the enzyme after digestion. Heating the reaction to 65°C for 20 min after digestion inactivates the majority of enzymes that have optimal incubation temperature of 37°C. Note that some restriction enzymes are not fully inactivated by heat treatment.