DNA clean-up
Fragment isolation
Competent cells
Probe Labeling
Probe Detection


In the Simple Cloning Lab, transformants are E.coli clones, harbouring a pGT4 plasmid, or a pGT4 derived plasmid. By a heatshock, the plasmids has been transferred into competent bacterial cells. As a result cells are ampicillin resistant, so they can be selected by growing on the antibiotic ampicillin.
In addition, we call them recombinants, when the plasmid that they harbour, is recombinant, i.e. having an insert between the BamH I and EcoR I sites of the plasmid. The insert can be a fragment of Lambda DNA (pGTλclones) or a few basepairs only, a result of filling in the BamH I site (pGT4ΔB clone). Since the BamH I site is in the tetracyclin resistance gene, recombinants among the transformants are tetracyclin sensitive, because the gene is disrupted.
(See also the pages pGT4 and pGTλclones in SCLResources)
The tetracyclin sensitivity can be used to select for recombinants among the transformants (negative selection: the ones that you're interested in, will not grow..).

Still, in one experimental step, the recombinants among a number of randomly selected transformants can be selected and grown, by inoculation of small liquid cultures in two 24-wells plates. Both (ampicillin medium containing) plates are inoculated with the same transformants, but one of the two plates contains additionally a second antibiotic: tetracyclin. So, recombinants can be identified because they will not grow in the tetracyclin wells, and the corresponding ampicillin cultures can be used for (recombinant) plasmid purification and subsequent agarose gel analysis.

  See the Movie about
Selection of Recombinants

Read the Movie Text

In this movie you will hear about (the formerly used) plasmid vector pBR322. This vector allows exactly the same transformants- and recombinants selection as pGT4 (see also the page pGT4 in SCLResources to compare the two vectors).