Transformation of E.coli
is part of the protocol to obtain bacterial clones,
or to "clone foreign DNA fragments into E.coli by using a plasmid vector".
cells, made competent by the treatment with calcium
chloride (or another chemical treatment), can be transformed by
uptake of foreign DNA. The transformation is a result of mixing the
DNA and the competent cells together, followed by a short heatshock; the
rapid temperature change creates a thermal imbalance on either side of
the E.coli membrane, which is thought to create a draft that sweeps
plasmids into the cell.
In the Simple Cloning Lab, E.coli transformation is typically done with the following solutions available:
The plasmid that is to be constructed, is formed by in vitro ligating the DNA fragments (plasmid vector fragments and donor fragments)
Transformation of the E.coli cells is accomplished by mixing
(at low temperature) plasmid DNA (or plasmids formed during a ligation)
with a small volume of a dense suspension of chemically treated E.coli
cells. A 90 seconds heatshock at 42°C will bring some of the DNA
molecules into the bacterial cells. After a one hour recovery at 37°C
the bacteria are spread on an agar plate which typically contains the
antibiotic for which the plasmid gives resistance. Transformants
(bacteria which successfully have taken up an intact plasmid molecule)
will be selected in this way, since non-transformants are not able to
grow on the antibiotic. Only circular DNA molecules, able to replicate,
may confer antibiotic resistance to the bacteria.
In the Simple Cloning Lab, for one cloning experiment, four transformations are done using the following DNA mixtures:
2, 3 and 4 are controls for the ligation and transformation protocol.
After one night at 37°C colonies are counted on the agar plates.