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 20l on 0.2-1.5g 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 2l or less of the DNA sample in a 20l 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 restr
