11: Restriction mapping

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Restriction endonucleases (REs) recognize and cleave specific sites in DNA molecules. In nature, REs are part of bacterial defense systems. In the molecular biology lab, they are an indispensable tool for both analyzing and constructing DNA molecules. In this lab, you will use REs to distinguish which plasmids contain S. cerevisiae or S. pombe ORFs or the bacterial lacZ gene.

Objectives

At the end of this lab, students will be able to:

describe the biological origins and functions of REs.
use online tools to identify recognition sites for REs in a DNA molecule.
devise a strategy to distinguish DNA molecules by selecting REs to use in DNA digests.
interpret the patterns of restriction fragments separated on agarose gels.

In the last experiment, your group isolated three different plasmids from transformed bacteria. One of the three plasmids, carries the S. cerevisiae MET gene that has been inactivated in your yeast strain. This MET gene was cloned into the pBG1805 plasmid (Gelperin et al., 2005). A second plasmid carries the S. pombe homolog for the MET gene, cloned into the the pYES2.1 plasmid. The third plasmid is a negative control that contains the bacterial lacZ⁺ gene cloned into pYES2.1. In this lab, your team will design and carry out a strategy to distinguish between the plasmids using restriction endonucleases. In the next lab, you will separate the products of the restriction digests, or restriction fragments, by agarose gel electrophoresis, generating a restric- tion map.