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included angle of 120° with an initial switch time of 5 s and a
final switch time of 45 s at 10 °C for 22 h on a CHEF II PFGE
machine (Bio-Rad). These parameters are ideal for separating
DNA of sizes of about 15 kb to approximately 1 mega base.
Ethidium bromide is added to the gel running buffer (20 μL
of 10 mg/mL solution).- Plasmids are visualized after gel running on a UV transillumi-
nator and photographed using a UVP geldoc II imaging sys-
tem (UVP Cambridge, UK). Plasmid number and sizes are
determined by comparison with the molecular size marker. - Gels are dried by placing overnight in a drying cabinet at 50C
between two sheets of blotting paper (see Note 6). Once the
gels are dried they can be stored for extended lengths of time
(>1 year) in a cool dry place between the pieces of blotting
paper. - Once dried, gels are rehydrated by placing in 200 mL of deion-
ized DNase-free water in a flat-bottomed pyrex glass bowl for
5 min (see Notes 7 and 8 ). If the gel is physically stuck to one
of the pieces of blotting paper, add the gel and blotting paper
to the water. After 5 min the gel is easily pulled away from the
blotting paper without damage. - The distilled water is discarded and 200 mL of denaturing
solution is added to denature the DNA in the gel. This is incu-
bated at room temperature for 45 min (see Note 9). - The denaturing solution is then removed and the gel neutral-
ized by addition of 200 mL of neutralizing solution and incu-
bated for 45 min at room temperature (see Note 9). - The neutralizing solution is removed and the gel placed in a
hybridization tube. 20 mL of prehybridization solution is
added to block the gel prior to probing. The gel is incubated
for at least 24 h at 65 °C (see Note 10). - Probes are made by first amplifying a desired gene by PCR
using specific primers and a bacterial strain that contains the
desired gene (In our example a resistance gene such as blaCTX-
M- 15 ) (see^ Note 11). - The probe is prepared by the random priming labeling method
using the purified PCR product prepared above as a template
(200 ng in 15 μL water Note 12 ) and radio-active P^32 dCTP
as a label. We use a commercially available kit using the stan-
dard protocol provided with the kit (see Note 13). - Once the probe has been labeled, unincorporated dCTP^32 and
unlabeled nucleotides are removed using a Sephadex G50
gravity flow gel filtration column: Briefly the labeled probe
3.2 In-Gel
Hybridization
Mark A. Toleman