Agriculture Reference
In-Depth Information
5.2.3 TOTAL COMMUNITY (TC) DNA
TC DNA was extracted from 0.5 g of soil after a harsh lysis step (FastPrep
FP120 bead beating system, MP Biomedicals, Santa Ana, Carlsbad, CA,
USA) by means of the BIO-101 DNA spin kit for soil (Q-Biogene). The
DNA was purified using the Geneclean Spin Kit (Q-Biogene) according
to the manufacturer's instructions. Purified TC DNA was stored at −20°C.
5.2.4 PCR AMPLIFICATION OF 16S RRNA GENE FRAGMENTS
AND DGGE ANALYSIS
Primer sets and PCR conditions employed in this study to amplify
Bac-
teria
[28],
Actinobacteria
[28], and
Alpha-
and
Betaproteobacteria
[29],
[30] and relevant information is provided in Table S1. DGGE of the 16S
rRNA gene amplicons was performed according to Gomes et al. [31]. The
gel was silver-stained according to Heuer et al. [32]. DGGE profiles were
analyzed by GelCompar 4.5. Dendrograms were constructed by means of
unweighted pair group method using arithmetic averages (UPGMA) based
on pairwise Pearson correlation indices, which were also subjected to per-
mutation tests with a modified version of PERMTEST software [33]. Box-
Whisker plots were generated using R (http://www.R-project.org) based
on dissimilarities (1- Pearson correlation indices) between samples within
the same treatment.
5.2.5 PCR AMPLIFICATION OF 16S RRNA GENES AND
PHYLOCHIP ANALYSIS
TC DNA extracts from three replicates per site and cover were amplified
using universal 16S rRNA gene primers (27f 5′- AGAGTTTGATCCTG-
GCTCAG-3′; 1492r 5′- GGTTACCTTGTTACGACTT-3′) and an 8-tem-
perature gradient PCR. At each temperature, approximately 5 ng of TC
DNA was used in 25 μl reactions (final concentrations were 1× Ex Taq
Buffer with 2 mM MgCl
2
, 300 nM each primer (27 f and 1492 r), 200
μM each dNTP (TaKaRa), 25 μg bovine serum albumin (Roche Applied
