131- 32 P labeled dCTP (EasyTides Deoxycytidine 5′-triphosphate.
(alpha^32 P), PerkinElmer, London, UK). - Unincorporated nucleotides are removed using a Sephadex
G-50 gel filtration column (illustra™ Nick™ Columns Sephadex
G-50 DNA grade, GE Healthcare Life Sciences, Little
Chalfont, Buckinghamshire, UK). - Detection film used is: Lumi-Film Chemiluminescent
Detection film, (Roche, Mannheim, Germany).
3 Methods
- Agarose plugs of bacterial strains are prepared using two differ-
ent previously published methods: For nonfermentative bacte-
ria such as Pseudomonas aeruginosa and Acinetobacter baumanii
we use a 2 day protocol [ 3 ]; For enteric bacteria such as E. coli
and Klebsiella pneumoniae we use the standard operating pro-
cedure for pulseNet PFGE of E. coli 0157, H7, Escherichia coli
non-0157 (STEC), Salmonella serotypes, Shigella sonnei, and
Shigella flexneri. Available on the Centres of Disease Control
website: CDC.gov using the web-link Disease Control web-
site: CDC.gov [ 4 ]. - Once the plugs are prepared they are digested with S1 enzyme
(see Note 1). Each individual plug is first washed at room tem-
perature for 20 min (see Note 2) in 1 mL of 1/10 TE buffer,
followed by a second incubation at room temperature for
20 min in 1 mL of 1× S1 buffer. Once the second wash is com-
pleted the S1 buffer is removed and the S1 enzyme is added.
The S1 enzyme is used at a very dilute concentration. To 6 mL
of 1× S1 buffer, 0.5 μL (50 U) of S1 (stock enzyme at a con-
centration of 100 U/μL) is added. Two-hundred microliters
of the enzyme solution is then used to digest each plug such
that the 6 mL is enough to digest 30 plugs. The digest is incu-
bated at 37 °C for 45 min. Once digested the plug is removed
from the digest solution and cut in half such that each plug is
enough for duplicate samples/gels. - Using the gel forming equipment provided by the supplier of
the PFGE system, 0.9% agarose gels are poured by adding 1 g
of PFGE grade agarose to 110 mL of 0.5× TBE buffer (45 mM
Tris, 45 mM boric acid, 1 mM EDTA) and microwaving at full
power for about 2 min (see Note 3). Gels are left to set for
about 20 min before plugs are loaded on the gel. - After digestion (see Note 4) each plug is cut in half, and half a
plug of each strain added to the gel(s) (see Note 5). Gels are
then inserted into the PFGE equipment under the gel running
buffer 2 L of 0.5× TBE Gel running parameter are 6v/cm; an
3.1 S1 Pulsed Field
Gel Electrophoresis
S1 Plasmid Analysis