Biochemistry 2280A Lecture : Restriction Enzymes are Enzymes
29 Dec 2011
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Like any other enzyme each restriction enzyme has preferred conditions in which it functions. Linear/constant relationship over time (positive) will plateau eventually when they are all cleaved. (%dna digested over time). Will reseal compatible sticky ends and much less efficiently blunt ends. Digest ~100ng of yfg and a plasmid vector with the same restriction enzyme or on that gives compatible overhanging sequences. Ligase forms covalent bonds (phosphodiester bonds) to seal the ends: need to get the plasmid into e. coli. E. coli have to be treated with chemicals to do it well. Mix e. coli with ligated dna; some of the e. coli will take up the plasmids (be. Transformed ): plate cells onto solid media (agar) containing antibiotic (ampilicillin). Single ells that have taken up the plasmid will grow, forming visible colonies. (blue) Not all of the transformed bacteria contain a plasmid that has yfg inserted.
