Extraction of total community DNA from biofilm pieces was performed using the alternative protocol in the UltraCleanTM Soil DNA Isolation kit (MoBIO Laboratories, CA, USA). In brief, 10 mg washed biofilm was first transferred into the supplied DNA extraction tubes. As an aid to homogenization of the material, the tube contents were mixed for 10 min at 250 rpm prior to the DNA isolation steps. The concentration of the cleaned DNA was measured with a nanodrop (Saveen Werner, Sweden) and the DNA was stored at −80 °C until use.
Ultraclean soil dna isolation kit
Manufactured by Qiagen
Sourced in United States
The UltraClean Soil DNA Isolation Kit is a lab equipment product designed for the extraction and purification of DNA from soil samples. The kit provides a straightforward process for isolating high-quality genomic DNA from a variety of soil types.
Automatically generated - may contain errors
Lab products found in correlation
Bead solution tubes, by Qiagen (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Miseq sequencing system, by Illumina (2 mentions)
Miseq platform, by Illumina (2 mentions)
Infinite 200 pro nanoquant, by Tecan (2 mentions)
454 gs flx, by Roche (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Silverstar taq dna polymerase, by Eurogentec (1 mentions)
Mgcl2, by Eurogentec (1 mentions)
Cellulose nitrate filter, by Merck Group (1 mentions)
S220 instrument, by Covaris (1 mentions)
Nextflex 96 dna barcodes, by PSC Biotech (1 mentions)
Membrane filter, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Myseq platform, by Illumina (1 mentions)
Mr dna, by Illumina (1 mentions)
44 protocols using ultraclean soil dna isolation kit
1
Biofilm Community DNA Extraction
2
16S rRNA Gene Amplification and Sequencing
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DNA was extracted using the UltraCleanTM Soil DNA isolation kit (MoBio Laboratories, Inc., Carlsbad, CA, United States) and amplified by PCR using the 16S rRNA gene universal eubacterial primers E338F (5′-ACTCCTACGGGAGGCAGCAGT-3′) and E797R (5′-GGGTATCTAATCCTG-3′) (amplicon length of 459-bp covering the variable V3 region of the 16S rRNA gene). The PCR mixtures were prepared with 2.5 μl 10x PCR buffer (Eurogentec, Seraing, Belgium), 1 μl MgCl2 (50 mM; Eurogentec), 2.5 μl dNTPs (2 mM; Thermo Scientific, Waltham, MA, United States), 1.0 μl of each primer (20 pmol μl-1 each; Eurogentec), 0.2 μl Silverstar Taq DNA polymerase (5.0 U μl-1; Eurogentec) and 1 μl standardized template DNA (10.74 ng μl-1) in a total reaction volume of 25 μL. The PCR was performed with the follow temperature/time conditions: initial denaturation at 94°C for 2 min, 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, and a final extension for 5 min (72°C). PCR products were evaluated on gel electrophoresis, the bands then were excised and purification was performed using a Qiaquick Gel extraction kit (Qiagen, Venlo, Netherlands). For the quantification of the PCR products, we used the PicoGreen dsDNA Assay Kit (Life Technologies, United States). Sequencing was performed at the Genomics Core of KU Leuven (Belgium) using a Roche 454 GS FLX+ (Switzerland).
3
Soil DNA Extraction and Bacterial Community Analysis
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DNA was extracted at the end of the experiment using an UltraClean TM Soil DNA Isolation kit (MO Bio Laboratories Inc., CA, USA). The extracted DNA was quantified by spectrophotometry (NanoDrop ® ND-1000) and its quality examined by electrophoresis on 0.7% agarose gel. The DNA extracts were then stored at -20°C for further analyses. We used ARISA (automated ribosomal intergenic spacer analysis) for our analysis of bacterial communities because of the relative ease, cost-effectiveness and reproducibility of the method. ARISA is a fast method to visualize the taxon richness of a biofilm. In the case of biofilms with low taxon richness, cloning was performed to identify the species dominating the biofilms.
4
Genomic DNA Extraction from Nitrifying Enrichments
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After incubation, the total volume of nitrifying enrichments resulting positive reaction for ammonia or nitrite oxidation and 2 L water samples were filtered on 0.22 μm pore size cellulose nitrate filters (Millipore, Billerica, USA). From samples concentrated on membranes, genomic DNA was extracted using Ultraclean TM Soil DNA Isolation Kit (Mo Bio Laboratories, Inc., USA) according to the manufacturer's protocol, supplemented by physical cell disruption. The membranes were shaken in "Bead Solution tubes" (Mo Bio Laboratories) containing microbeads at 25 Hz for 2 min with cell mill MM301 (Retsch, Haan, Germany) to enhance cell disruption.
5
Soil DNA Extraction and Shotgun Library Preparation
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The total genomic DNA of pellets was extracted using the UltraClean® Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA), according to the manufacturer’s instructions. The purified DNA was resuspended in 200 μL of DNase/RNase-free water and kept at −20 °C, until their use for sequencing. The concentration and quality of total DNA were assessed using NanoDrop ND-1000 (Nanodrop Technologies, Thermo Scientific, Wilmington, DE, USA) and agarose gel electrophoresis. Sterilized water samples were used as a negative control for DNA extraction, and the PCR results showed that the negative control water samples did not yield detectable 16S rRNA products.
For construction of the shotgun library, approximately 5 μg of the DNA sample was mechanically sheared to 350 base-pair fragments, using a Covaris S220 instrument (Covaris, Woburn, MA, USA). Libraries were constructed using the Apollo 324 Next Generation Library Preparation System (IntegenX, Pleasanton, CA, USA) with the NEXTflex-96 DNA barcodes (Bioo Scientific, Austin, TX, USA).
For construction of the shotgun library, approximately 5 μg of the DNA sample was mechanically sheared to 350 base-pair fragments, using a Covaris S220 instrument (Covaris, Woburn, MA, USA). Libraries were constructed using the Apollo 324 Next Generation Library Preparation System (IntegenX, Pleasanton, CA, USA) with the NEXTflex-96 DNA barcodes (Bioo Scientific, Austin, TX, USA).
6
Biomass Extraction from Sponge Cubes
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Three sponge cubes were sampled individually from both the top and bottom sampling ports (Fig. 1 ), and referred to as U and B samples subsequently in this study. New replacement sponges were placed back into the reactor after each biomass sampling so as to provide new substrata for the adherent bacteria. Subsequent samplings were carried out such as to avoid sampling the replacement sponges. Sponges with the adherent biomass were placed in 50 mL centrifuge tubes, each containing 20 mL of 1X phosphate buffer saline (PBS). The samples were then vortexed at maximum speed for 10 min and the sponge cubes removed aseptically by sterile forceps. The biomass suspensions in the 50 mL tubes were then centrifuged at 6800×g for 10 min to collect cell pellets. 0.2 g of biomass from cell pellet was weighed and extracted for its DNA. DNA was extracted using the UltraClean Soil DNA Isolation Kit (MoBio Laboratories, Carlsbad, USA) with slight modifications to the protocol by adding lysozyme and achromopeptidase to the lysis buffer [21 (link)].
7
Monitoring Reactor Performance Metrics
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To monitor reactor performance, mixed liquor and effluent samples were collected, filtered through a membrane filter (0.45 μm; Whatman, Maidstone, United Kingdom), and analyzed for acetate, PO43−-P, NH4+-N, NO3−-N, and NO2−-N. The concentrations of PO43−-P were determined according to standard methods (65 ). Total ammonia (NH3 + NH4+) concentrations were analyzed using the salicylate method (method 10031; Hach Company, Loveland, CO). Acetate, nitrite, and nitrate were measured using high-pressure liquid chromatography as previously described (9 (link)).
Seven milliliters of biomass samples from the reactors was collected weekly and stored in 15% glycerol at −80°C until DNA extraction was performed. DNA was extracted using the UltraClean soil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA). Extracted DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and stored at −80°C.
Seven milliliters of biomass samples from the reactors was collected weekly and stored in 15% glycerol at −80°C until DNA extraction was performed. DNA was extracted using the UltraClean soil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA). Extracted DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and stored at −80°C.
8
Bacterial 16S rDNA Profiling via MiSeq
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Bacterial DNA was extracted from the GF using an Ultra Clean Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, California, USA) in accordance with the manufacturer's instruction. The hypervariable V3-V4 region of 16S rDNA was amplified by PCR with 341f7 (link) and R8068 (link) primers. PCR was performed in accordance with the method reported by Takahashi et al.9 (link) Sequencing was conducted using a paired-end and modified to 2×300 bp cycle run on an Illumina MiSeq sequencing system (Illumina, San Diego, California, USA) and MiSeq Reagent Kit V.3 chemistry.
9
Environmental Microbiome DNA Extraction
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Genomic DNA was extracted from 1 g of each environmental sample (mixture of water and sediments for the river samples and solid material for the terraces samples) or 2 mL of each enrichment culture using Ultraclean Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA), following the manufacturer’s instructions. DNA samples were visualised on a 1% (w/v) agarose gel with ethidium bromide staining and quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA). Amplicon sequencing was performed via Molecular Research LP Mr DNA (Shallowater, TX, USA) in the Illumina MySeq platform, targeting the V3–V4 region of the prokaryotic 16S rRNA gene (341F-CCTACGGGNGGCWGCAG- and 785R-GACTACHVGGGTATCTAATCC-primers) for the six environmental samples and the four enrichment cultures and the Internal Transcribed Spacer region of the rRNA genes (ITS1F-CTTGGTCATTTAGAGGAAGTAA- and ITS2R-GCTGCGTTCTTCATCGATGC-primers) was chosen for fungi detection for the six environmental samples and the two heterotrophic cultures.
10
Profiling Microbial Diversity in Sediment
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DNA from 14N incubation samples #3731 T0d, #3731 T326d, and #5133 T170d was extracted from ~0.5 g of sediment using the UltraClean Soil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA) and microbial community composition was profiled using the 515f (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806r (5′-GGACTACNVGGGTWTCTAAT-3′) primer pair (Caporaso et al., 2011 (link)). PCR barcoding reactions were conducted in house at Caltech and amplicons were outsourced to Laragen Inc. (Laragen Inc., Culver City, CA) for sequencing on the Illumina MiSeq platform. Resulting sequence data consisted of 250 bp paired end reads, which were assembled into a single contig and analyzed using Qiime 1.8 (Caporaso et al., 2010 (link)). Sequences were trimmed to a minimum quality value of 30, and taxonomy was assigned to each OTU representative based on the SILVA 115 rRNA database uclust (Edgar et al., 2011 (link)) using a sequence similarity threshold of 99%. The full protocol is contained in a GNU makefile (Supplemental Data Sheet 1); the makefile must be in the same directory as the raw sequencing reads, and qiime 1.8 and usearch must be installed. The complete protocol can be run by typing “./orphanlab_itag_protocol.mk all” into the command prompt.
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