Bio-Rad Ligation and Transformation Module - Manual (Page 28)

Process of bacterial transformation. Competent E. coli are transformed with plasmid DNA.
Only a few bacteria take up the plasmid DNA. Bacteria are then plated on selective media and
incubated overnight. Only bacteria that contain the plasmid will grow and form colonies. Bacteria
colonies are then picked and grown for use in plasmid minipreps.
Minipreps of Plasmid DNA
Once a plasmid has been introduced into competent bacterial cells and
the cells have grown into colonies on a medium selective for cells containing
the plasmid, the next step is to prepare a miniprep of the plasmid DNA in
preparation for sequencing or further experiments. The three steps are: 1)
growing cells in liquid culture; 2) purifying the plasmid DNA from the culture;
and 3) performing a restriction digest on the purified DNA to determine
whether the DNA insert in the plasmid is the expected size. To start the
liquid culture, cells from an isolated bacterial colony are placed in selective
medium (nutrient broth with an antibiotic to which the plasmid provides
resistance). This placing of the cells into the medium is called inoculation.
It is important to choose a single isolated colony from the plate, so that the
liquid culture will contain cells that all have the same plasmid. If cells from
more than one colony are used for inoculation, the miniprep may contain
multiple plasmids and the mixed DNA will not be useful for sequencing or
further experiments.
Restriction digestion of plasmid DNA
Before proceeding with further experiments using the purified plasmid
DNA, it is important to verify that the isolated plasmid contains the insert
of interest using restriction digestion followed by gel electrophoresis.
Plasmid DNA
Transformation
Competent E. coli
Transformed cell
Transfer to
selective media
Incubate at
37°C overnight
Pick colonies
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