Environmental Engineering Reference
In-Depth Information
matographed over a column packed with Al
2
O
3
to obtain an apolar fraction
using hexane/dichloromethane (9:1, v/v) as an eluent and analyzed by gas chro-
matography (GC) and GC-mass spectrometry (GC-MS) as described elsewhere
[11].
2.5 Total Organic Carbon (TOC)
The total organic carbon content was determined by elemental analysis (EA)
isotope ratio monitoring mass spectrometry (EA/irmMS) as described by Coo-
len [11].
2.6 Extraction of Total DNA
Total DNA was extracted from 0.25 g of sediment using the UltraClean Soil
DNA Kit following the descriptions of the manufacturer (Mobio, Carlsbad,
CA, USA). Because our study relied on the analysis of 16S rDNA by PCR
amplification, it was of utmost importance to prevent any contamination of
the sediment samples by foreign DNA. Extensive precautions against conta-
mination of the samples were applied as described previously [10, 13]. As a
control for contamination during DNA extraction, a parallel sample without
sediment was subjected to the whole extraction and purification procedure (ex-
traction control). All DNA extracts were tested for the presence of co-extracted
impurities resulting in polymerase chain reaction (PCR) inhibition [11, 12].
The same extraction method was applied to the POM collected on the filters.
Prior to extraction, the filters were sliced with a sterile scalpel. The total DNA-
extract for each sediment sample and POM sample was quantified with the
fluorescent dye PicoGreen (MoBiTec, Gottingen, Germany).
2.7 Amplification of 16S rDNA
Partial 16S rDNA of GSB within the POM and Holocene sediment layers
of Ace Lake were amplified by PCR in a Geneamp PCR System 2400 (Perkin
Elmer, Connecticut, USA). 16S rDNA solely found in GSB were selectively
amplified using primer 341f (5'-cct acg gga ggc agc ag-3' [33]) in combination
with gsb840r (5'-atg acc aac atc tag tat t-3' [34]). A 40-bp-long GC-clamp
(5'-cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc c-3' [33]) was attached
to the 5'-end of primer 341f to prevent complete melting of the PCR products
during denaturing gradient gel electrophoresis (DGGE). The reaction mixtures
were prepared according to Coolen [12]. The PCR conditions were: Initial
denaturing at 96
◦
C (4 min) followed by 35 cycles including denaturing at 94
◦
C
(30 sec), primer annealing at 57
◦
C (40 sec), and primer extension at 72
◦
C. A
final extension step was performed at 72
◦
C (10 min). Each PCR amplification
series included one reaction without DNA template, which served as a control
for contaminations during the pipetting of the reaction mixture components. A
