Hi christo,
The recombinant DNA technology or simply molecular cloning consists on the preparation of identical copies of a recombinant DNA (vector + DNA insert) transformed into cell host generally a bacteria. starting with the creation of complementary ends of vector and DNA insert (here is the role of two different endonucleases or enzymes of restriction) and the ligase enzyme will do the ligation between them. many types of restriction enzymes exist and the choice of the right one is important.
https://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/restriction_enzymes/index.html
Best Regards!
Dear Farah Fadwa Ben belgacem Mam, Can you discuss about the types of restriction enzymes and the choice to be made ?
Different restriction enzymes have specificity for different DNA sequences, so you would choose a restriction enzyme that cuts a vector open at one site, and digests either end of your PCR-amplified insert, after which they can be ligated together. Because both ends of your insert would be complimentary to either end of your vector, of the successful ligations approximately 50% will be in the correct orientation, and after transforming into competent cells, you should isolate the constructs from a few colonies and perform restriction digests on them to confirm you have a clone with the insert in the correct orientation in the vector i.e. predict the sizes of the DNA fragments in a gel based on the restriction enzymes you are using, and see if that is what turns up in the gel.
Dear Benedict Macintyre Sir, Can you discuss about the specificity of different restriction enzymes for different DNA sequences ?
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