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sample. An appropriate volume was loaded depending on the expected DNA
concentration, although it was usually between 5 and 50 µl. The gel was usually run at
80V/cm (power supply 500/400 = Amersham Pharmacia Biotech, Davy Avenue,
Knowlhill, Milton Keynes, MK5 8PH, United Kingdom) for 30 min or until the dye had
migrated approximately three quarters of the length of the gel. However, this varied (as
stated above) depending on the samples were being observed.
Following electrophoresis, the gel was placed in the ImageMaster® VDS unit
(Pharmacia Biotech) to be examined under a ultra-violet transilluminator and
photographed using a video camera attached to the system and a computer, so that digital
image could be taken.
2.9.3 -
Amplified ribosomal DNA restriction analysis
2.9.3.1 -
PCR amplification of 16S rRNA gene
The following procedure was adapted from Vaneechoutte et al. (1995) (ARDRA
methodology outlined in figure 2.2). Bacterial specimens were grown in 30m1 glass
universal bottles in 10ml of Tryptic Soya Broth (Sigma). Of this culture, 1 µl was added
to a 49µl PCR reaction mixture consisting of 21 µl sterile RO H2O, 16µ1 dNTP mixture
(Abgene, 98 College Avenue, Rochester, New York 14607, United States) (6.26µl of
each dNTP made up to I ml in sterile H20), 5µl Buffer IV (ABgene), 4p1 25mM MgCl
(ABgene), 1µl universal primer 1.5 F (5'-TGG CTC AGA TTG AAC GCT GGC G-3')
(Sigma-Genosys, 1442, Lake Front Circle, The Woodlands, Tx, 77380), 1 µl universal
primer 1.5 R (5'-TAC CTT GTT ACG ACT TCA CCC CA-3') (Sigma-Genosys)
(Vaneechoutte et al., 1995), 1 µl Taq DNA Polymerase (ABgene).
Both primers were diluted 1: 5 from their original supplied concentrations with
sterile reverse osmosis water. Taq DNA polymerase was only added after `hotstart' on
the thermal cycler or PCR block (Techne Progene, Techne (Cambridge) Ltd, Duxford,
Cambridge, CB2 4PZ, England). Total volume of PCR reaction mixture with DNA
template was 50µ1. Mixture was run on the PCR block using the following program.
Initial denaturing step of 10 min at 95°C followed by the addition of ice cold Taq (1µl per
reaction). This is followed by 30 cycles of 0.5 min at 95°C (denature), 1 min at 55°C
(anneal), 1.5 min at 72°C (extension), after which there was a single cycle of 72°C for 8
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