Restriction Enzymes and Agarose Gel Electrophoresis.docx

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John Coleman

Restriction Enzymes and Agarose Gel Electrophoresis Restriction Enzymes and Vectors-What are they and how do we use them?  Bacteria can produce DNases, which can double cleave the DNA at specific sites  Restriction endonucleases or restriction enzymes can cleave the DNA at specific sites  The 1 endonuclease to be identified is EcoRI isolated from E.Coli  Many other restriction enzymes have been isolated  Restriction enzymes can provide a defense system for DNA against viruses and bacteriophages  Restriction enzymes remember different restriction sites  Majority of Restriction enzymes recognize either 4 or 6 base pairs  EX. EcoRI recognizes and cleaves GAATTC, HaeIII recognizes and cleaves GGCC  Restriction maps can be used to determine where the enzymes will cleave  Restriction enzymes play a key role in DNA cloning The Basic Steps required to clone a gene are:  1. The fragment of DNA containing the gene to be cloned is inserted, with restriction enzymes and ligation enzymes into a vector producing recombinant DNA. Vector is usually circular but it can also be linear.  2. The vector acts as a vehicle to transport the gene into a host cell, which is often a bacterium.  3. Host cell multiples producing multiple copies of the vector.  4. The recombinant DNA is replicated.  5. After a large number of cell divisions a colony can be produced Two naturally occurring DNA  1. Small cir
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