Restriction Endonuclease Digestion of a Plasmid

 

Tested Studies in Laboratory Teaching, 2008, Volume 29

Margaret M. Dooley

Abstract

Recombinant DNA technology is widely utilized and therefore, undergraduate laboratory courses incorporate these techniques. This reliable laboratory exercise introduces the student to plasmid vectors, restriction enzymes, and agarose gel electrophoresis. Restriction digestion of pBR325 with PstI and HindIII, in single and double digests, was performed and analyzed on an agarose gel. Students constructed a standard curve based on the migration of fragments from a HindIII digest of bacteriophage λ DNA and used that curve to estimate the sizes of DNA fragments in the pBR325 digests. In this exercise, measurements on the largest λ fragment were included to illustrate the resolution limits of agarose gels and the proper use of standard curves.

Keywords:  restriction enzymes, agarose gel electrophoresis, phage, recombinant DNA technology

University of Kentucky (2007)