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

RNA 41F (5'-GCT CAG ATT GAA CGC TGG CG-3') and 16S rRNA 1066R (5'-ACA
TTT CAC AAC ACG AGC TG-3'), which amplified the 1025 bp fragment of the 16S
rRNA gene which contained the V3 region. Amplification of a smaller fragment (i. e. not
the whole gene) limited the possible amplification of non-specific products during PCR.
The PCR reaction for the amplification of the V3 region was as follows: To 3 pl
of genomic DNA the following reagents were added, 2.5 µl of PCR buffer (Gibco BRL),
1.25 µl MgCl (Gibco BRL), 0.5 µl of dNTP mix (Gibco, BRL), 1 µl of primer V3F (5'-
CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG
CAG CAG-3' with GC clamp; Invitrogen), 1 µl of primer V3R (5'-ATT ACC CGC GCT
GCT GG-3'; Invitrogen) (primers taken from Muyzer et al., 1993) and 2.5 pl of Taq
polymerase (Invitrogen). This was made up to 25 µl. The reaction mix was placed in 0.25
µl thin walled micro-Eppendorf tubes and positioned in a PCR block (Techne Progene).
The following `step-down' thermal cyclic program was taken from Ercolini et al.
(2001 a): 1 cycle of 5 min at 94°C; 10 cycles of 94°C (1 min), 66.0 -
56.0 (1 min) and
72°C (3 min); 20 cycles of 94°C (1 min), 56°C (1 min), 72°C (3 min); and finally one
cycle of 72°C (10 minutes) to ensure complete extension of all strands. The V3 fragment
was 233 bp long. The reaction was checked by gel electrophoresis of 2µl of PCR product
(Section 2.9.2).
2.9.5.4 -
Denaturing gradient gel electrophoresis
DGGE was performed using the D-code system apparatus (Bio-Rad, Bio-Rad
Laboratories Ltd., Bio-Rad House, Maylands Avenue, Hemel Hempstead, Hertfordshire
HP2 7TD) using a method from Muyzer et al. (1993). The PCR products were run in 8%
(wt/vol) polyacrylamide gels in 1X TAE (Sambrook et al., 1989) with denaturing
gradients formed with 8% (wt/vol) acrylamide stock solutions (acrylamide-N, N' -
methylenebisacrylamide
37: 1) containg 0% and 100% denaturants (7 M urea (Sigma)
and 40% (vol/vol) deionised formamide (Sigma). Gels were formed by the setting of a
1.5 mL 8% polyacrylamide solution with 0% denaturant as a base layer, upon which the
15 mL denaturing gradient gel was poured to produce a 30%-50% gel. Electrophoresis
consisted of 50 V voltage for 10 min which allowed the DNA to migrate slowly out of
the wells. Then electrophoresis was run at 140 V for 6 h. After electrophoresis, the gels
were stained for 3 min in ethidium bromide (0.5 mg L-1) and then rinsed for 15 minutes in
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