DNA from the biofilms of PE and glass, as well as seawater communities, was extracted using the DNeasy PowerBiofilm kit (Qiagen) according to the manufacturer’s instructions, which included a bead-beating step. DNA was quantified using a Qubit® HS DNA kit (Life Technologies Corporation) and samples were diluted to equalize the concentration. PCR amplifications were performed using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs® inc.) and the primer pair 515F-Y and 926R ([44 (link), 45 (link)], Supplementary Table S1 ), which amplified regions V4-5 of the 16S rRNA gene of bacteria, using PCR conditions as described previously [45 (link)]. PCR products were purified with Ampliclean magnetic beads (Nimagen, The Netherlands). Index PCR was performed using Illumina Nextera Index Kit v2 adapters. Sample normalization was done with the SequelPrep™ Normalisation Plate Kit (ThermoFisher Scientific) and samples were pooled for sequencing. Pooled libraries were quantified using the NEBNext Library Quant Kit for Illumina (New England Biolabs, UK) and diluted to 4 nM. Negative DNA extraction controls and negative controls for sequencing were processed simultaneously.
Dneasy powerbiofilm kit
Manufactured by Qiagen
Sourced in Germany, United States
The DNeasy PowerBiofilm Kit is a laboratory product designed for the extraction and purification of DNA from biofilm samples. It provides a standardized and efficient method for isolating genomic DNA from complex microbial communities found in biofilms.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (4 mentions)
Qubit hs dna kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Dneasy powersoil kit, by Qiagen (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Qubit 3, by Thermo Fisher Scientific (2 mentions)
Double stranded dna dsdna high sensitivity assay kit, by Thermo Fisher Scientific (2 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Fastprep, by MP Biomedicals (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Dneasy powerwater kit, by Qiagen (2 mentions)
Nextera kit series, by Illumina (2 mentions)
Q5 hot start high fidelity 2x master mix, by New England Biolabs (1 mentions)
Nextera index kit v2 adapters, by Illumina (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Colibri microvolume spectrophotometer, by Berthold Technologies (1 mentions)
Q5000 spectrophotometer, by Quawell (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Powerlyzer 24 homogenizer, by MP Biomedicals (1 mentions)
Hotstartaq plus master mix kit, by Qiagen (1 mentions)
48 protocols using dneasy powerbiofilm kit
1
Standardized 16S rRNA Gene Amplification and Sequencing
2
Microbial Community Profiling of Biofilms
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DNA was extracted at the end of each experiment (GDM: day 30, enrichments: day 15) from 20 whole membranes of the GDM biofilms and from the remaining volume (∼80 mL) of 12 samples of the enrichment experiment using the DNeasy PowerBiofilm Kit (Qiagen, Germany). The extraction was largely performed according to the manufacturer’s specifications, except for the inhibitors removal step that was extended to 1 hour. The purified DNA was stored at -20°C in 10 mM Tris buffer for further analysis. DNA was amplified with the primer pair 799F-1115R that exclude chloroplasts/cyanobacteria (Chelius and Triplett, 2001 (link); Redford et al., 2010 (link)); the reverse primer was modified according to Silva et al. (2018) (link). Partial 16S rRNA gene sequences were obtained using an Illumina MiSeq platform (LGC Genomics, Germany). Raw data processing, the definition of operational taxonomic units (OTUs), and their taxonomic assignment were performed by an in-house pipeline as described previously (Silva et al., 2018 (link)). Raw sequencing data have been deposited on the Sequence Read Archive of NCBI under the project number PRJNA529182.
3
DNA Extraction from Biofilm Samples
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For French samples, 2 ml of biofilm was centrifuged at 13,000 rpm for 30 min at 4°C, the supernatant containing ethanol was removed and the pellet used as starter for DNA extraction. Samples were then extracted using the DNA extraction kit Macheray‐Nagel NucleoSpin Soil kit following manufacturer recommendation starting with SL1 solution and with a final elution of 30 µl with the solution provided in the kit, as described in Vasselon, et al. (2017 (link)). For Swiss samples, DNA extraction was performed using 1 ml of the preserved sample. After centrifugation at 3'500 g during 5 min, the supernatant containing ethanol was removed and the pellet used as starter for DNA extraction. Each sample was extracted using the DNeasy PowerBiofilm kit (Qiagen) according to the manufacturer's instructions, as described in Apothéloz‐Perret‐Gentil et al. (2017 (link)) in a final elution of DNA in 100 µl with the solution provided in the kit. All DNA extracts were then stored at –20°C until PCR amplifications.
4
Microbial Mat Genomic DNA Extraction
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Genomic DNA of the microbial mats was extracted by using DNeasy PowerBiofilm Kit (QIAGEN, Hilden, Germany) following manufacturer's instructions. Genomic DNA of each microbial mat consisted in two separated extractions of 0.5 g combined at the elution step to increase DNA concentration for sequencing analysis. A negative control of the kit was also performed. DNA concentrations were determined in a NanoDrop ND 1000 spectrophotometer (Thermo Fisher Scientific) and stored at −20°C until sequencing analysis.
5
Metagenomic Analysis of Subgingival Microbiome
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The subgingival microbial DNA in a 500 µL transport medium was extracted by using the DNeasy PowerBiofilm kit (Qiagen, Hilden, Germany) with a preceding bead beating (45 s; speed: 3450 oscillations/min) and stored at −20 °C. Then, the DNA concentration was determined by a Colibri Microvolume spectrophotometer (Titertek Berthold, Pforzheim, Germany). The 16S rRNA gene of each sample was amplified using the primer pairs 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) targeting the V3–V4 region. Library preparation was achieved by the Illumina MiSeq platform generating 300 bp paired-end reads. The raw sequence data were imported into QIIME2 [51 (link)], in which paired-end reads were merged and denoised into amplicon sequence variants (ASVs) using the DADA2 plugin [52 (link)]. The reads were filtered based on exact matches to the barcode/primer and an average quality score of 30. Due to the issue of sample bleeding between the Illumina MiSeq runs [53 (link)], the low-abundance filters (a cutoff threshold of 0.1% of mean frequency 70,651) were applied to reduce the number of spurious ASVs in the data set.
6
Genomic DNA Extraction from Biofilms
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Genomic DNA for sequencing analysis was obtained from biofilms grown in the AAA model. Biofilms were harvested by transferring the glass coverslips into 5 mL of sterile PBS. Then, they were sonicated for 15 minutes to disperse the biomass. Genomic DNA extraction was done using the “DNeasy PowerBiofilm Kit” (Qiagen, Germantown, US), following the manufacturer’s instructions. Briefly, biofilms were subjected to chemical and mechanical lysis. Proteins and inhibitors were removed, followed by pH-driven precipitation of large insoluble macromolecules. Total genomic DNA was captured using a silica spin filter column and finally eluted. DNA concentration was measured using a NanoDrop (Thermo Scientific).
7
Metagenomic DNA Extraction from Biofilms
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Metagenomic DNA extraction was done using “DNeasy PowerBiofilm Kit” (Qiagen) following the manufacturer’s instructions. DNA concentration was measured using UV-Vis Spectrophotometer Q5000 (Quawell).
8
Macroalgal Biofilm and Coral Tissue Extraction
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Prior to extraction, the macroalgal biofilm was separated from the algal tissue by overnight incubation at 200 rpm in 10 mL 1x PBS at 37 °C. Coral fragments were defrosted on ice and the tissue was stripped from the skeleton with an airgun into 1x PBS solution, homogenised for 1 min at 12.5 rpm with a tissue homogeniser, pelleted (10 min at 16,000 rcf) and snap frozen in liquid nitrogen prior to DNA extraction. DNA from seawater, sediment, sponge and macroalgal biofilms was extracted with the DNeasy PowerSoil kit (Qiagen) and DNA of coral tissue and mucus samples was extracted using the DNeasy PowerBiofilm kit (Qiagen) following the Manufacturer’s instructions. DNA extracts were stored at − 80 °C until being sent for sequencing.
9
DNA Extraction from Biofilm Samples
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Genomic DNA (gDNA) was extracted using the DNeasy PowerBiofilm Kit (QIAGEN). The samples were transferred from −80 ˚C to room temperature. In a laminar flow hood, each biofilm swab sample was placed into a PowerBiofilm Bead Tube using sterile forceps. Qiagen's protocol (DNeasy® November 2016) was followed according to the manufacturer's instructions, except the first step was omitted, since the saturated biofilm material was attached to the swab; therefore, no weighing and centrifugation was applicable. To bead‐beat, the sample, a PowerLyzer 24 Homogenizer (MP Biomedical, FastPrep‐24™ 5G) was used. At the final step, extracted DNA was eluted following the manufacturer's instructions and stored in −80 ˚C. The quantity and partial quality of nucleic acid samples were assessed based on absorbance spectrums using a spectrophotometer (Thermo Scientific, NanoDrop 1000).
10
Extraction of Community DNA from Biofilm
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Extraction of the total community DNA from the initial biofilm sample (0.5 g) and the enrichment cultures of each month was carried out by using the DNeasy® PowerBiofilm Kit (Qiagen, Germany) following the instructions of the manufacturer. 40 ml of the enrichment cultures were centrifuged at 2360 g for 15 min using a Rotanta 460 R centrifuge (Hettich, Germany), and the community DNA was extracted from the pellet.
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