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.

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 [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.

• 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

• 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).

Never make up restriction digests with the restriction enzyme composing more than 1/10 of the final volume. This is due to the fact that the restriction enzyme proteins are stored in glycerol. At concentrations above 10%, glycerol not only inhibits the digestion but also can cause star activity - leading to aberrant, non-specific cuts of the DNA. Set up a control digest when using restriction enzymes. This helps you know if the enzyme is working properly and you set the reaction up well. Use a plasmid DNA which generates known fragmentation patterns. When preparing double enzyme digests, determine the salt content of the buffers. Conduct restriction digestion using the enzyme with the lower salt requirements FIRST, then use the second enzyme by adjusting the reaction tube's buffer to the second optimal salt concentration. DNA preparations may have impurities which can inhibit restriction enzyme digestion activity. Some DNA isolation kits even use high EDTA buffers to elute the DNA. This is ok for DNA storage, but high concentrations of EDTA can inhibit restriction digestion. PCR purify your DNA and elute with TE or water if your enzyme activity is inhibited but your DNA looks ok. You could also add Mg2+ to the reaction and see if that helps also. If you cannot obtain complete restriction digestion of your DNA after adding extra enzyme, set up a new digest and add spermidine to a final concentration of 2 mM.

Safety

The safety implication of the procedure.