Transformation

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

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".

E.coli 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 general, bigger DNAs give lower transformation efficiencies (that is why students only rarely find a pGTλ15721 clone in the Lambda library..).
Typically, the selection of cells that have actually taken up the DNA is done by growing them on agar plates containing a selective antibiotic.

In the Simple Cloning Lab, E.coli transformation is typically done with the following solutions available:

  • competent E.coli cells, freshly made or from -80 stock
  • a ligation reaction mixture
  • a DNA digest (as used in the ligation reaction)
  • a dilute solution of a control plasmid

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 42C will bring some of the DNA molecules into the bacterial cells. After a one hour recovery at 37C 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.
Linear DNA will not replicate (and will not survive exonuclease  activities) inside the bacterial cell!

In the Simple Cloning Lab, for one cloning experiment, four transformations are done using the following DNA mixtures:

  1. ligation reaction mixture
  2. vector DNA digest and donor DNA digest (a mixture of the two, as used in the ligation reaction)
  3. 1 ng of uncut plasmid DNA (e.g. the vector plasmid)
  4. water ( = no DNA)
     

2, 3 and 4 are controls for the ligation and transformation protocol.
In a standard heat shock transformation the one ng plasmid in 3 should give 100-1000 colonies.

After one night at 37C colonies are counted on the agar plates.
Each colony represent one single clone, which means, that all bacteria in the colony are identical, since they have grown from a single transformed bacterial cell.
Transformation of E.coli using one nanogram of a plasmid with a size of a few kilobasepairs results in 100 to 1000 transformants on the agar plate.