Team:Cambridge/Protocols/Restriction Enzyme Digestion

Restriction Enzyme Digestion

A method that allows to create a restriction map of a given DNA fragment. Widely used to test for the correct integration of a cloned sequence into a vector.

Theory

How it works

Practice

• Master mix preparation

2.0μl of DNA

0.5μl of each restriction enzyme (or the respective amount of water for the control with uncut plasmids)

0.1μl of BSA - acetylated Bovine Serum Albumin enhances the performance of restriction enzymes

5.9μl of water

1.0μl of 2×NEBuffer

• Check for the compatibility of restriction enzymes chosen

To check if the two selected restriction enzymes can perform effective catalysis in the same solution, go to the website of New England Biolabs → select Double Digest Finder → choose which restriction enzymes you want to use → follow the digest recommendations.

• Procedure

1. incubate at 37°C for 2 hours
2. perform gel electrophoresis (10μl of DNA-restriction enzyme mixture in each well)
3. compare with predicted fragment sizes
4. remember about molecular weight markers

Safety

The safety implication of the procedure.