However, by choosing an enzyme or enzymes that will cut your plasmid into multiple fragments, you can get a very unique pattern that will distinguish one 5kb backbone with a 1.2kb insert from all others. A useful restriction enzyme based technique for verifying plasmids like this is "plasmid fingerprinting", where you cut the plasmid into 3-8 pieces such that all (or most) fragments are small enough to be accurately sized on a gel and also such that they are different enough in size to be easily resolved from each other. This is particularly true if you receive a plasmid from someone in another lab, or dig one out of the freezer and you are not 100% sure it is what you are looking for, but you have a map and know exactly what it should be. Often, it will be enough to know that you have a 1,200bp insert in a 5,000bp backbone, but there are many plasmids out there that, when digested with restriction enzymes common to multiple cloning sites, will result in similar sized bands, thus making this simple digest less informative. Many DNA analysis tools, includingĪddgene’s Sequence Analyzer, allow you to identify which restriction sites are present in a given sequence.įor a list of the commonly used commercially available restriction enzymes, see By selecting the appropriate enzyme(s), one can either linearize a plasmid to determine the size of the entire construct or excise some or all of an insert from it.īefore beginning your diagnostic digest, you will need to select a restriction enzyme or enzymes that cut your plasmid. The pattern of the fragments on the gel can indicate if the plasmid contains the expected size insert. The goal of a diagnostic digest is to cut your plasmid into specific sized pieces and analyze the resulting fragments by
Often, the size of the plasmid insert and vector backbone are known and thus this technique can be quickly used to verify your plasmid. A diagnostic restriction enzyme digest takes advantage of the fact that restriction enzymes cleave DNA at specific sequences called restrictions sites. Given the variety of these enzymes and the unique sites they recognize, restriction digests have become the most widely used method scientists employ to selectively move a specific piece of DNA from one plasmid to another. Restriction enzymes are naturally occurring bacterial endonucleases that recognize a large range of DNA sequences.
0 kommentar(er)
