What is an analytical restriction enzyme reaction?

An analytical restriction enzyme reaction, also known as a single digestion, is usually performed in a volume of approximately 20µl on 0.2-1.5µg of substrate DNA using 2 to 10-fold excess of enzyme over DNA (2-10 units per microgram DNA). To prevent the possibility of aberrant results, the volumes of DNA and enzyme used should not exceed certain levels. For example, if a volume of DNA >10% of the reaction volume is used, there is the potential that any contaminants (including EDTA) present in the DNA sample could inhibit the digestion reaction. Therefore, we recommend using 2µl or less of the DNA sample in a 20µl reaction. Similarly, we recommend limiting the volume of enzyme to 10% or less of the reaction volume to limit the final glycerol concentration to 5%. Higher glycerol concentrations can result in star activity (the digestion of sequences similar to, but not identical to, their normal recognition sites) with certain enzymes.

The following is an example of a typical analytical restriction enzyme reaction. Components are added in the order listed to a sterile microcentrifuge tube:   

*The Acetylated BSA supplied with Promega’s restriction enzymes is 10mg/ml, and therefore, should be diluted 1:10 with Nuclease-Free Water prior to use. Alternatively, the 10mg/ml BSA may be diluted 1:100 directly into the restriction enzyme digestion.

Mix gently by pipetting (do not vortex once enzyme is present), close the tube and centrifuge briefly at 12,000 x g in a microcentrifuge to collect the contents at the bottom of the tube. Incubate at the optimum temperature for 1-4 hours. (Although the optimal temperature for the majority of restriciton enzymes is 37°C, it can vary.) Add 4µl of Blue/Orange Loading Dye, 6X, and proceed to gel analysis.

Please see Table 3 (Composition of Promega Restriction Enzyme Buffers [1X]) of Restriction Enzyme Properties for salt compositions and pH of different restriction enzyme buffers. See Table 2 (Relative Activity of Restriction Enzymes in Promega's 10X Buffers) for the relative activity of restriction enzymes in Promega’s core restriction enzyme buffers at the appropriate incubation temperatures.