Sureclean plus

An equal amount of each PCR product (n = 230 per reservoir) was pooled into a single multiplexed sample (one such sample per reservoir), immediately purified using SureClean Plus (Bioline) according to the manufacturer's protocol, then stored at −20°C until sequencing. PCR products were stored in separate freezer compartments from DNA extracts to minimize the risk of cross-contamination. One sequencing library per multiplexed sample was prepared using a TruSeq Nano DNA Library Preparation Kit (Illumina) according to the manufacturer's protocol except that only six enrichment PCR cycles were performed. Sequencing was performed on a HiSeq 2500 system (Illumina) using the HiSeq Rapid SBS Kit v2 in Rapid Run Mode, to obtain paired 250 bp reads.

Biofilm samples were taken with a sterile cannula directly from the electrode (n = 2 for each reactor). Genomic DNA was extracted with the NuceloSpin Tissue Kit (Macherey-Nagel, Germany). The microbial community composition on DNA level was analysed with a standard TRFLP procedure using the primers UniBac27f (FAM labelled) and Univ1492r for amplifying the partial sequence of the 16S rRNA gene of bacteria [54 ].
The PCR Master Mix contained 6.25 μL enzyme mix (MyTaq HS Red Mix, 2x,Bioline, Germany), 0.25 μL of each primer (5 ρmol μL⁻¹, MWG Biotech, Germany), 4.75 μL nuclease-free water, and 1 μL genomic DNA (about 360–720 ng). The PCR cycle parameters were as follows: 1 min at 95°C, 25 cycles of 15 s at 95°C, 15 s at 54°C, and 2 min at 72°C, followed by a 10 min final extension step at 72°C [55 (link)]. PCR products were purified (Sure Clean Plus, Bioline) and digested with restriction endonucleases HaeIII and RsaI (New England Biolabs GmbH, Germany). After product precipitation (EDTA/EtOH), TRFLP analysis was performed using an ABI PRISM Genetic Analyzer 3130xl (Applied Biosystems, USA) and MapMarker® 1000 (BioVentures Inc., USA) as size standard.

DNA was extracted from the filters or the Eppendorf tubes using the phenol/chloroform-based method described by Zwirglmaier et al. [48 (link)]. Subsequently, DNA was purified using Sure Clean Plus (Bioline, London, UK) and eluted in sterile ultrapure water. The DNA was then stored frozen until further analysis.

DNA was extracted from the filters or the Eppendorf tubes using a phenol/chloroform-based method described by Zwirglmaier et al. [49 (link)]. Subsequently DNA was purified using Sure Clean Plus (Bioline, London, UK) and eluted in sterile ultrapure water. The DNA was then stored frozen at −80 °C until further analysis.

To isolate a region of L1 targeted by the LA-HPV and the Abbott Real-Time PCR method, conventional PCR for HPV DNA viral amplification of a ~ 450 bp HPV-specific segment from the L1 gene covering nucleotide positions (5722-6162) numbered according to NC001526 HPV-16 reference genome was performed using the forward primer MY09 5′-CGTCCMARRGGAWACTGATC-3′ and the reverse primer MY11 5′-GCMCAGGGWCATAAYAATGG-3′. Five microliters of DNA were added to 15 μL of reaction mix containing 1 × PCR buffer, 0.2 mM dNTPs, 4 mM MgCl₂, 0.3 μΜ of each primer, and 2 U/μL of Platinum Taq DNA Polymerase, High fidelity (Invitrogen, USA).
The thermocycling conditions were denaturing at 96 °C for 10 s, annealing at 50 °C for 5 s, and final extension at 60 °C for 4 min for 25 cycles. PCR products were subjected to electrophoresis in 4% agarose (Applichem) using 1 × TBE buffer (Applichem) and visualized under UV light. PCR products were then purified using commercially available SureClean Plus (Bioline) and the purified products were sequenced directly via automated sequencing using two overlapping PCR primers (both forward and reverse). The BigDye Terminator v3.0 kit (Applied Biosystems; Foster City, CA, USA) was used for sequencing using the automated Sequencer (ABI PRISM 3130xl; Applied Biosystems).