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Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR |
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.
note:
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).
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