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DNA Cloning

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Biochemistry 2288A
Chris Brandl

DNA CloningMost applications of recombinant dna tech require relatively large amounts of your favorite gene YGF One way to get this ammount of DNA is to clone it Cloning of YGF is usually accomplished by having it replicated in EColi Second way is to do PCRPlasmid fragment of dna that replicates independently from the host chromosome Key is independent replicationEcoli chromosome one copy 3 million bp Big circular dna one copy percellPlasmid dnacopy number varies depeding on palsmit from 1100 copiescell 3000bp most plasmids that we work with are circular Key features that makes plasmid useful for cloning 1Origin of replciation since it has to replciate independently of host 2A selectable marker usually a gene encoding resistance to an antibiotic eg ampicillin tetracycline 3A useful restriction site into which YGF can be inserted much useful if there is only one in the plasmidand it also cant be in origin or selectable marker The three key features First step cut YFG with restriction enzyme Leave frament with 2 sticky endsDigest 100ng of YFG and a plasmid vector with the same restriciton enzyme or one that gives compatible ov
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