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 [http://biologylabs.utah.edu/jorgensen/wayned/ape/ 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 [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.