| Comments |
The table shows
combinations of priming sequences
and templates, which researchers have used for
gene replacements. A colony of a bacterial
strain bearing the transposon can serve
as template in PCR with primers that
amplify the cassette and have 40-base
flanks targeting the cassette to a site
in the chromosome of a recipient. The
PCR product in each case is small (1–2
kb) and is introduced via electroporation
into a Red-expressing recipient
strain, in which it generates a recombinant
that can be easily selected. That
is, the recombinant forms a reasonably
large colony on a plate of LB agar
containing a high enough concentration
of an antibiotic to suppress colony
formation by 108 nonrecombinants,
following overnight incubation at 37°C.
The bla gene of Tn3 also works well
for this purpose, but is perhaps less
useful due to its widespread use as a
selectable marker (ampicillin resistance)
on plasmid cloning vectors. |