Fig. 1. Subcloning of a DNA fragment, in this case containing a putative promoter fragment, from one clone into another vector. Judicious selection of an appropriate restriction enzyme yields a recombinant plasmid containing the inserted promoter fragment in the recombinant plasmid. Drug resistance markers are located on each of the plasmids, allowing selection of the appropriate plasmid after bacterial transformation. The plasmids contain enough markers so that it is easy to discriminate among the three plasmids shown in the figure. The expression vector contains the β-lactamase gene, which allows bacteria containing this plasmid to grow in the presence of ampicillin. This plasmid also contains the gene for β-galactosidase. In the presence of a substrate called X-gal, colonies containing this gene will turn blue. The restriction enzyme (RE) site for RE #1 is within this gene, and when a fragment is successfully cloned into this site, the gene is interrupted and is no longer capable of functioning to convert the substrate, X-gal, into the blue product. Thus, colonies with the insert in the expression vector will be white. Bacterial colonies which are TetR and AmpR (tetracycline resistant and ampicillin resistant, respectively) result from the original plasmid containing the promoter fragment. Neither of the other plasmids (the expression vector or the recombinant vector) contain the TetR gene, and they will be TetS (sensitive to tetracycline), so colonies containing these plasmids will fail to grow on plates containing tetracycline. To select the appropriate colonies, we plate the initial transformants on Amp plates containing X-gal. One day later we will pick the white colonies. We will replate them on Amp plates in an ordered pattern and on Tet plates in the same ordered pattern. Those that are AmpR, white, and TetS will be the colonies that contain the appropriate plasmid. We will verify the legitimacy of the plasmid by preparing a small amount of the plasmid from these colonies and digest the plasmid with RE #1, the restriction enzyme that we used to make the construct. Valid constructs should give us the two identically sized fragments that we used to make the recombinant plasmid.