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everse osmosis water and photographed using the ImageMaster® VDS unit (Pharmacia
Biotech) with UV transillumination
(302nm). Position of bands was analysed using the
ImageMaster 1D elite V3.01 gel analysis software (Amersham Pharmacia Biotech &
Non-Linear Dynamics) as shown for ARDRA analysis (section 2.9.4).
2.9.6 -
Sequencing of bacterial 16S rDNA
For each isolate of interest the 16S rRNA gene was amplified using PCR (section
2.9.3.1) and the nucleotide sequence of the gene was determined using chain-termination
sequencing (Sanger et al., 1977) performed at the biopolymer synthesis and analysis unit
(BSAU) Nottingham University. For most of the isolates, sequencing was performed
directly on the purified PCR product; however, in some cases, this did not produce
satisfactory results and the product was cloned using the TOPO TA Cloning reaction
(Invitrogen).
2.9.6.1 -Purification of 16S rDNA PCR products
It was necessary to purify the 16s rDNA PCR product prior to sequencing to
remove any primers, excess salts and other contaminants (enzymes, unincorporated
nucleotides, agarose dyes, etc. ) which could interfere with the sequencing reaction. The
QlAquickTM PCR purification kit (Qiagen) was used to purify the PCR amplicons. The
following information is modified from the QlAquickTM handbook (Qiagen). The kit used
a system in which nucleic acids were absorbed to a silica-gel membrane while the
contaminants were passed through during microcentrifugation.
The QlAquick PCR
purification protocol was as follows: 5 volumes of buffer PB were added to one volume
of PCR product, the solution was then mixed. The solution was added to a QlAquick spin
column that was placed inside a 2m1 collection tube. This was centrifuged at 10,000 g
(13,000 rpm, Biofuge pico, Heraeus) for 1 min. The flow-through was discarded and
0.75m1 of PE buffer (wash buffer) was added to the spin column, which was placed back
in the empty 2ml collection tube. This was then centrifuged (13,000 rpm, 1 min). The
flow-through was discarded and the spin column was centrifuged for one minute to
remove the entire wash buffer. The column was then placed in a 1.5m1 Eppendorf tube
and 30µl of elution buffer (sterile reverse osmosis water) was added to the spin column
(directly onto the membrane) and allowed to stand for 1 min. It was then centrifuged for
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