Experiment 1: Restriction Digestion

What is Restriction Digestion?

Restriction Digestion is the process of cutting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases (sometimes just called Restriction Enzymes or RE's). These special enzymes recognize specific sequences in the DNA molecule (for example GATATC) wherever that sequence occurs in the DNA.

Restriction Digests begin by mixing the DNA and the RE, but it's unfortunately not quite as simple as that. Restriction Enzymes are delicate and need to be treated carefully. Because enzymes are proteins and proteins denature as the temperature is increased, RE's are always stored in a freezer until they are used. In fact, all of the ingredients in a Restriction Digest are kept on ice until it's time for the reaction to begin. The actual reaction conditions vary from one enzyme to the next, and include temperature, NaCl and/or MgCl₂ concentration, pH, etc. All of these variables except temperature are optimized by mixing the enzyme and DNA with a buffer specific for the enzyme of choice. Once all the ingredients are mixed in the reaction tube, the tube is incubated at the Restriction Enzyme's optimal temperature for 1 hour or longer. Then finally when the digest has run for the appropriate amount of time, the reaction tube is put back on ice to prevent nonspecific degradation of your DNA. Once the Restriction Digest is completed, Agarose Gel Electrophoresis is performed to separate the digest fragments by size and visualize the fragments and perhaps purify them for further experiments.

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