Bio-Rad Ligation and Transformation Module - Manual (Page 24)

Possible ligation products.
Transformation
Once a gene or part of a gene has been amplified using PCR and ligated
into a plasmid, the next step in cloning is transformation, introducing the
plasmid into living bacterial cells so that it can be replicated. Heat shock
transformation and electroporation are the two methods of bacterial
transformation commonly used in the laboratory. Both methods require
competent cells, bacterial cells that can take up DNA. Not all cells are
naturally competent. For example some species, such as
Bacillus subtilis,
can be easily transformed, but for other species, such as Escherichia coli,
only a small number of cells in a culture may be able to take up DNA.
Competent cells may be prepared in the laboratory or purchased
commercially.
PCR Fragment
PCR Fragment
PCR Fragment PCR Fragment
PCR Fragment
PCR Fragment
20
No interruption of
lethal gene so
transformed
bacteria will die
Can be minimized
by controlling
molar ratio of
inserts
Product cannot
replicate
Desired product –
insert can be in
either orientation
Self-ligation
of vector
Multiple
inserts
Self-ligation
of inserts
Ligation
of vector
and insert