Environmental Engineering Reference
In-Depth Information
second reaction with 1 µl of the extraction control was amplified by PCR as
a control for contamination during the extraction of DNA from the sediment
samples. A third reaction containing 15 ng of DNA of
Flavobacterium
sp. was
used to monitor the specificity of the PCR reactions.
2.8 Denaturing Gradient Gel Electrophoresis (DGGE)
All PCR-products were separated by DGGE [33]. DGGE was carried out in
a Bio-Rad D Code system (Bio-Rad). All PCR products were separated based
on their variations in the nucleotide positions on 6% (wt/vol) polyacryl amide
gels which contained a 20
70% linear gradient of denaturant. Electrophoresis
proceeded for 5 h at 200 V and 60
◦
Cin1
−
TAE pH 8.3. After ethidium
bromide staining, DGGE-bands were cut out with a sterile scalpel and rinsed
with ultra pure water (Sigma, USA). The DNA of each band was eluted [11, 12].
One µl of the eluted 16S rDNAs were re-amplified, using the primers for
GSB, in order to generate template DNA for the subsequent cycle sequencing
reactions.
×
2.9 Sequencing of DGGE Bands
Primers and dNTPs were removed from the re-amplified DGGE bands using
the QIAquick PCR Purification Spin Kit (Qiagen) and 10 ng of DNA was
subjected to cycle sequencing reactions using the conditions as described by
Coolen [11, 12].
2.10 Phylogenetic Analysis
Sequence data were compiled using ARB software [31] and aligned with
complete length sequences of closest relatives obtained from the databases of
the ribosomal database project II (RDP-II, [32]) and GenBank [1] using the
ARB FastAligner utility. Matrices of similarity, distance and phylogenetically
corrected distance values were generated using the maximum parsimony option
in ARB. Sequences obtained in this study have been deposited in the GenBank
sequence database under accession numbers AY303358 and AY665401.
2.11 Real Time Quantitative PCR
Real time quantitative PCR was performed in an iCycler system (Biorad,
Hercules, CA, USA) in order to quantify the amount of DNA derived from
green sulfur bacteria in the POM samples of the extant water column as well as
the 42 Holocene sediment layers of Ace Lake. The method was slightly modi-
fied after Coolen et al. (2004b). To quantify DNA of GSB, the PCR conditions
and primers (without GC-clamp) were used as described above. Accumulation
of newly amplified rDNA was followed online (80
◦
C for 25 sec) as the increase
