ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

Molecular Weight (Fig 2.6b), three 100bp ladders were run with the restriction digests in
the gel and were used to standardise MW across the gel. This standardisation allowed
comparison between gels. Fifthly, Band matching (Fig. 2.7), where bands were matched
to themselves and common bands in a computer generated reference lane that held all the
standardised values for the bands in the gel being analysed. This provided data on
similarities between lanes in relation to the position of bands, which allowed phenetic
similarity calculations between the patterns based on band position.
This procedure was employed to produce a dendrogram showing the phenetic
relatedness of the AFP active isolates ARDRA patterns. The data were then transferred
to ImageMaster 1D database V2.12 (Amersham Pharmacia Biotech) to calculate a
phenetic dendrogram created from the comparison of all the bands from all the lanes from
more than one gel. The ARDRA patterns were matched by Rf value using the Dice
similarity coefficient (Equation 2.1) and were displayed in a UPGMA (Unweighted pair
group method with arithmetic averages) clustered dendrogram.
SD =
2a
2a+b+c
Equation 2.1 -
Dice Coefficient equation. SD = coefficient of similarity
based on the Dice equation; a
= the number of positive matches. b and c= the number of non-matching characters between pairs of
Operational Taxonomic Units (Priest and Austin, 1995).
2.9.5 -
Denaturing gradient gel electrophoresis
2.9.5.1- Collection of bacterial community
To obtain a community fingerprint it was necessary to obtain a concentration of
the bacterial community from each lake. This was performed by filtering 120mL of lake
water through a 47mm diameter, 0.2 µm mixed cellulose esters (nitrate and acetate) filter
(Millipore). The biologically inert mix of nitrate and acetate make the filters ideal for
cellular harvesting. The filter was stored in 1 ml of TE buffer in a 1.5m1 Eppendorf tube at
-70°C.
2.9.5.2 -
Genomic extraction for DGGE
To provide a clean DNA template for the initial PCR step in DGGE analysis the
DNA was extracted using a modified CTAB technique (Ausubel et al., 1999), which is
outlined here.
The stored filters in TE buffer were defrosted on ice. Once totally defrosted. the
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