Sputum samples were defrosted in batches and DNA was mechanically extracted using an MPBio FastPrep®-24 bench-top homogeniser and FastDNA™ SPIN Kits for Soil (MPBio http://www.mpbio.com/product.php?pid=116560200 ) in accordance with the manufacturer’s instructions.
Fastdna spin kits for soil
Manufactured by MP Biomedicals
Sourced in United States
The FastDNA™ SPIN Kits for Soil are a set of products designed for the rapid and efficient extraction of DNA from soil samples. The kits utilize a proprietary bead-beating technology to effectively lyse cells and release the genetic material, which is then purified and concentrated for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Fastprep 24, by MP Biomedicals (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Miseq pe300 platform, by Illumina (1 mentions)
Lib l kit, by Roche (1 mentions)
454 gs flx titanium system, by Roche (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
Miseq platform, by Illumina (1 mentions)
Taq pcr master mix, by Eppendorf (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Fluostar optima instrument, by BMG Labtech (1 mentions)
6 protocols using fastdna spin kits for soil
1
DNA Extraction from Sputum Samples
2
Quantifying Ammonia-Oxidizing Archaea and Bacteria in Environmental Samples
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Total DNA was extracted from 0.5 g of freeze-dried weathered rocks and sediments using the FastDNA® SPIN kits for soil (MP Biomedicals, United States). The concentration and quality of the extracted DNA were determined using a Nanodrop 2000 (ND2000, Thermo Fisher Scientific) spectrophotometer for subsequent experiments. AOA was amplified using the primer set of Arch-amoA26F/Arch-amoA417R (Park et al., 2008 (link)), and the primer set of amoA1F/amoA2R was used for AOB (Weiner and Maizels, 1999 (link)). Paired-end sequencing of the amoA functional genes of AOA and AOB was performed on the Illumina Miseq PE300 platform at Shanghai Personal Biotechnology, Co., Ltd., Shanghai, China. Quantification of the amoA genes of AOA and AOB was performed using the primer sets of Arch-amoAF/Arch-amoAR and amoA1F/amoA2R, respectively, with the systems and reactions as described previously (Rotthauwe et al., 1997 (link); Francis et al., 2005 (link); Gao et al., 2016 (link)). The R2 values of the standard curves were 0.95 or higher in this study. The abundance of each gene was normalized to the number of qPCR-derived gene copies per gram of dry weight sample. All raw sequence reads were deposited in National Omics Data Encyclopedia (NODE) with the project numbers OER444534 for AOA and OER445462 for AOB.
3
Sludge DNA Extraction and 16S Sequencing
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Prior to the RNA-SIP experiment, 5 ml of sludge samples were removed and stored at -20°C for molecular analysis as described in Ho et al. [21 (link)]. DNA extractions were performed using FastDNA Spin kits for Soil (MP Biomedicals, Australia) according to the manufacturer’s instructions. The concentrations of each eluted DNA were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technology, Rockland, DE). The extracted DNA was loaded on a 1% agarose gel to identify extent of DNA degradation. Genomic DNA was then subjected to 16S amplicon sequencing which is described below.
4
Bacterial DNA Extraction and Sequencing Protocol
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Samples for DNA analysis were kept at −80°C until DNA extraction was performed (in 2012). DNA was extracted from the samples using FastDNA® SPIN Kits for Soil and a FastPrep-24 bead-beading machine (MP Biomedicals, Santa Ana, USA), according to the manufacturer's instructions. Bacterial DNA was amplified using the barcoded primers 338F and 926R according to the protocol described in Torondel et al. (2016 (link)). PCR products were cleaned using the Wizard PCR product purification kit (Promega, Fitchburg, Wisconsin, USA) and were then pyrosequenced at the Wellcome Sanger Institute in 2012 using the Lib-L kit on the 454 GS FLX Titanium System (Roche, Branford, Connecticut, USA). Further details on the protocols used to generate 16S rRNA gene sequence data are as described in Torondel et al. (2016 (link)).
5
Bacterial Genomic DNA Extraction and 16S rRNA Amplification
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Total bacterial genomic DNA samples were extracted using the Fast DNA Spin Kits for soil (MP Biomedicals, Santa Ana, CA) according to the manufacturer's instructions. The completeness of the DNA sample was then assessed by 1% agarose gel electrophoresis. The V3 to V4 hypervariable region of the bacterial 16S rRNA gene was amplified with the primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACNNGGGTATCTAAT-3’) (Munyaka et al., 2015 ). The PCR was carried out on a Master cycler Gradient (Eppendorf, Germany) using 25 μL reaction volumes, containing 12.5 μL 2 × Taq PCR MasterMix, 3 μL BSA(2 ng/μL), 1 μL Forward Primer (5 μM),1 μL Reverse Primer(5 μM), 2 μL template DNA, and 5.5 μL ddH2O. Cycling parameters were 95°C for 5 min, followed by 28 cycles of 95°C for 45 s, 55°C for 50 s, and 72°C for 45 s with a final extension at 72°C for 10 min. The raw paired-end (PE ) reads data was obtained through the Illumina Miseq platform.
6
Groundwater DNA Extraction Protocol
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In the laboratory, DNA extractions for all groundwater were conducted using FastDNA spin kits for soil (MP Bio, USA) according to the manufacturer's manual. DNA yield was quantified based on spectral absorbance at 260 nm, ratios of 260/280 nm and 260/230 nm, respectively (Thermo Scientific NanoDrop, 2000) , and then stored at -80 °C until required for PCR amplification. The final DNA concentrations were quantified by PicoGreen, using a FLUO star Optima instrument (BMG Labtech, Jena, Germany).
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