DNA extraction
For the ethanol samples, DNA extractions were performed using the
Promega Wizard®SV Genomic DNA Purification System. One filter was cut
into six pieces and separately lysed overnight at 55°C in 275 µl lysis
buffer. The lysate of two pieces was pooled and the three pooled lysates
were processed separately using the manufacturer’s protocol. This
resulted in three DNA extracts of 50µl from one filter for each ethanol
sample, which were pooled together. Half of the pooled DNA extract (75
µl) underwent cleanup using the Promega Wizard® DNA Clean-Up System and
was eluted in 50µl TE-buffer following the manufacturer’s protocol.
For the bulk samples, specimens and ethanol were mixed into a
homogeneous solution using a blender or, in case less than 100 ml sample
was available, grinded with mortar and pestle. Depending on the volume
of the sample, two (for samples < 100 ml) or six (for samples
> 100 ml, this was the case for the three replicates of
station 120) subsamples of 2 ml were transferred to an Eppendorf tube
for DNA extraction. The remaining “soup” was stored at -20°C. The
Eppendorf tubes with the 2 ml “soup” were centrifuged and the
supernatant was removed. To remove the remaining ethanol, the Eppendorf
tubes were incubated at 50°C with open lid until the pellet was dry. The
pellet was then used to extract DNA using the DNeasy PowerSoil Kit
(QIAGEN) following the manufacturer’s protocol. The lysis step was done
by adding 10 µl of Proteinase K (20 mg/ml) and incubating overnight in a
thermal shaker at 56°C and 1000 rpm. The same volume of each subsample
was pooled to obtain an end volume of 50 µL, which was then cleaned with
the Wizard DNA Clean-Up System (Promega) according to manufacturer’s
protocol. From each of the 12 bulk samples, one DNA extract of 50 µL was
obtained.