Team:Cambridge/Protocols/RestrictionDigest

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 DNA (prepare a master mix that does not contain restriction enzymes).
2. Incubate at 37°C for 2 hours.
3. Perform gel electrophoresis (10μl of DNA-restriction enzyme mixture in each well). Remember about molecular weight markers that help to assess the size of separated DNA fragments.
4. Compare with predicted fragment sizes. You can create a restriction map using ApE - A plasmid Editor.

• Tips on Experimental Design

• Try (if possible) to use a restriction enzyme that will cut within the fragment that you have attempted to insert. If your fragment has not inserted, you should get a single band. This is a fool proof demonstration that your Gibson has not worked properly.
• Attempt to make the resulting fragments as small as possible, so you can get the greatest size resolution. Smaller fragment separate more than larger fragments.
• Do not use enzymes that will produce fragments of approximately the same size. These may become difficult to distinguish if there is any smearing of your bands.

• 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 New England Biolabs → select Double Digest Finder → choose which restriction enzymes you want to use → follow the digest recommendations.