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Restriction Digest

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Restriction enzymes are able to cut polypeptide chains at specific sites. Restriction digest is the process of using several different restriction enzymes to cut DNA into small pieces that can be sequenced. You are then able to line up the overlap in the different sequences to determine the complete sequence of the DNA. Restriction enzyme sites are used to make maps of DNA. Restriction enzymes provide a critical tool for molecular biologist to predictable fragment a given DNA molecule, which allows them to analyze the structure and features in fine detail.

In the 1960s Stuart Linn and Werner Arber found both modification enzyme and restriction nucleases in extracts of E. coli strain B. This explained why sometimes when E. coli was transfected …show more content…

An important part of this is which enzymes will work in the same buffer. Using multiple restriction enzymes is necessary because without multiple enzymes it would impossible to determine the specific sequence of the DNA strand. Once you have determine which restriction enzymes you want to use, you will then set up the digest. You will need several 1.5 mL tubes (at least 7 tubes if you are using 3 enzymes). In each tube you need to add some of your DNA sample. You will then add different combination of restriction enzymes to each tube. The typical order of combination of enzymes would be 3 tubes that have one enzyme each, 3 tubes that have 2 enzymes each and one tube that has all the enzymes. It is necessary to have different combinations of the restriction enzymes so that you get a better idea of how the enzymes cut in relation to each other. Then you will add buffer, BSA, and water. Once the tubes are mixed they have to be incubated so that the restriction enzymes have time to cut the DNA into fragments. After the incubation the tubes of DNA fragments are ran through a gel electrophoresis, which allows you to visualize the results of the

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