![]() ![]() Restriction endonucleases (RE), also referred to as restriction enzymes, are proteins that recognize short, specific (often palindromic) DNA sequences. Type II REs cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. ![]() Many restriction enzymes will not cut DNA that is methylated on one or both strands of the recognition site, while some require substrate methylation. Mix by pipetting and collect the contents at the bottom of the tube.įor a digestion with a single RE the reaction is very simple: Restriction digestion is one of the most common reactions performed in molecular biology. Incubate at the appropriate temperature and the appropriate time for the enzyme. Add 4µl of 6X Blue/Orange Loading Dye and analyze digested DNA by gel electrophoresis. Preparing an insert for transfer from one vector to another usually requires digestion with two different REs. If both restriction enzymes work in the same restriction enzyme buffer, the reaction is straightforward. Simply add 1µl of the second restriction enzyme and adjust the amount of water used to maintain a 20µl reaction. Remember, restriction enzymes are commonly stabilized in 50% glycerol solution. Do not exceed 5% glycerol in final digest with the two enzymes. Restriction Digestion: Other Considerationsĭo both enzymes work at the same temperature? Glycerol concentrations >5% may lead to enzyme star activity, which means the enzyme will lose sequence specificity and may produce random cuts in the DNA. The majority of restriction enzymes work best at 37☌, but those isolated from thermophilic bacteria require higher temperatures for maximal activity (e.g., BstXI and BstZI work best at 50☌). Some enzymes work best below 37☌, like SmaI (25☌) and CspI (30☌).
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