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

Restriction enzymes, also called restriction endonucleases, are enzymes naturally found in bacteria which serve as a protection against foreign DNA found in a cell which usually implies a phage infection. The enzymes introduce a double-strand cuts in the DNA leaving either blunt or sticky single-strand ends, recognizing specific, usually 4bp or 6bp palindromic sequences.

Practice

• Master mix preparation

For 2.0μl of DNA solution add:

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

• Procedure

1. Mix DNA solution with the suitable amount of the master mix. As a control, repeat the same procedure with uncut plasmid
2. incubate at 37°C for 2 hours
3. perform gel electrophoresis (10μl of DNA-restriction enzyme mixture in each well)
4. compare with predicted fragment sizes
5. remember about molecular weight markers

• Tips on Restriction Enzyme Usage

• Store restriction enzymes in the freezer, at around -20°C.
• Never make up restriction digests with the restriction enzyme composing more than 1/10 of the final volume. The reason is that restriction enzymes are stored in glycerol, and at concentrations above 10%, glycerol not only inhibits the digestion but also changes enzyme specificity (star activity).
• Check for the compatibility of the restriction enzymes chosen.

To check if the two selected restriction enzymes can perform effective catalysis in the same solution, go to the website of [http://www.neb.com New England Biolabs] → select [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp? Double Digest Finder] → choose which restriction enzymes you want to use → follow the digest recommendations.

Safety

The safety implication of the procedure.