133(60 μL) is added to the top of the gel filtration column fol-
lowed by 320 μL of 0.1 M Tris pH 7.5. The filtrate flows
through the column by gravity flow and is collected in a 1.5 mL
eppendorf tube and is discarded. A new eppendorf tube is then
placed under the column and the labeled DNA is eluted with
430 μL of 0.1 M Tris pH 7.5. This results in unincorporated
nucleotides being left on the column. The 430 μL of labeled
probe is then boiled for 6 min in a screw cap eppendorf tube
and then added to the prehybridized gel and left to hybridize
overnight at 65 °C (see Note 13).- Once hybridized the probe is discarded and the gel is washed
for 1 h at 65 °C with 2× SSC 0.1% SDS (100mls) and then
again for 1 h at 65 °C with 0.1× SSC, 0.1% SDS (100 mL) (see
Note 14). - Washed gels are finally removed from the hybridization tubes,
washed under a warm tap for a couple of minutes and blotted
dry with blotting paper. They are then wrapped in cling film
and placed against a sheet of film overnight in a film cassette
before developing using standard film development and fixer
solutions (see Note 15) (Fig. 1 ).
Fig. 1 (a) S1 pulsed field gel of 13 E. coli strains (lanes 1–13) run against a Saccharomyces cerevisiae molecu-
lar size standard (Lane 14) and illuminated with ethidium bromide staining under ultraviolet light. The chromo-
some of each bacterial isolate is the brightest band at the top of the gel and plasmids are visualized as bright
bands towards the lower end of the gel. Each E. coli isolate has between 1 (lane 11) and 6 (lane 2) plasmids.
(b) Auto radiograph of (a) after probing with a radioactive P^32 -labeled blaCTX-M-15 probe. Note the blaCTXM-15 gene
is found on the chromosome in E. coli isolates (lanes 2, 3, 5, 6, 9, and 12) on multiple plasmids in lanes 1 and
13 and on the chromosome as well as on an individual plasmids in lane 3 and 6. It is only found on the plasmid
in lanes 8, 10, and 11. Positive blaCTX-M-15 plasmids are highlighted in (a) with a blue circle
S1 Plasmid Analysis