Fast dna kit for soil

Genomic DNA was extracted using the Fast DNA Kit for Soil (MP Biomedicals, Solon, OH) according to the manufacturer’s protocol with two 45-s beat beating steps using a Fastprep Instrument (MP Biomedicals, Solon, OH). Afterwards, DNA was purified and concentrated using the DNA Clean & Concentrator kit (Zymo Research Corporation, Irvine, CA). The DNA concentrations were either determined with the NanoDrop® ND-2000 (Thermo Fisher Scientific, Waltham, MA) or the Qubit 2.0 fluorometer (Thermo Fisher Scientific).

Total microbial community DNA was extracted using FastDNA Kit for Soil (MP Biomedicals, Heidelberg, Germany) according to the manufacturer’s instructions. The quality of DNA was detected by NanoDrop™ OneC UV-Vis Spectrophotometer (Thermo, Waltham, MA, USA) to ensure the concentration and purity of the DNA. The primers 547F (5′-CCA GCA SCY GCG GTA ATT CC -3′) and V4R (5′ ACT TTC GTT CTT GAT YRA -3′) were used in the first PCR to amplify against variable region 4 (V4) of the 18S rRNA gene sequence. The PCR was performed by using a total volume of 25 μL containing 14.75 μL ultrapure water, 5 μL buffer [5×], 1 μL of each Forward and Reverse primer [10 μM], 0.25 μL Fast pfu DNA Polymerase [5 U/μL], 2 μL of dNTPs [2.5 mM], and 1 μL DNA template. The cycling program was set up with the following steps: high-temperature denaturation at 98 °C for 2 min; 20 cycles of denaturation at 95 °C, 30 s; annealing at 58 °C, 30 s; extension at 72 °C, 1 min; final extension at 72 °C, 5 min. PCR amplicons were purified with VAHTS^TM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Equimolar DNA concentrations of each amplicon were then sent for paired-end Illumina MiSeq sequencing (read length: 2 × 250 bp) to Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

The ΔrecA mutant strain (KS3) was generated using “redirect” PCR targeting (55 (link), 56 (link)). The recA coding sequence (vnz_26845) on the cosmid vector Pl1-B2 (http://strepdb.streptomyces.org.uk/) was replaced by an oriT-containing apramycin resistance cassette, which was amplified from pIJ773 using the primer pair ks6 and ks7. The resulting disrupted cosmid (pKS100) was introduced into S. venezuelae by conjugation via E. coli ET12567/pUZ8002. The resulting exconjugants were screened for double-crossover events (Apr^r Kan^s), and deletion of recA was verified by PCR analysis using the primers ks8, ks9, and ks10. A representative recA null mutant was designated KS3. For complementation, plasmid pKS2 (P_recA-recA) was introduced into the ΔrecA mutant by conjugation.
Genomic DNA for genome sequencing of the ΔrecA mutant (KS3) and the parental wild-type strain was performed using the FastDNA kit for soil (MP Biomedicals Germany). Genomic DNA of biological replicate samples per strain was sequenced by MiGS (Pittsburgh, USA).

The reactor sludge was sampled from the bottom of the reactor at the end of phase I (stage I), stage I, and stage II. The samples were immediately fixed with 100% ethanol at a ratio of 1:1 (v/v) and stored at −20 °C before DNA extraction. Total DNA was extracted using a Fast DNA Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) following the manufacturer’s instructions. The extracted DNA was then subjected to quality inspection using a UV-Vis spectrophotometer (NanoDrop® ND-1000, Agilent, Santa Clara, CA, USA).

Genomic DNA was extracted from all triplicate incubations at the beginning of the experiment and after 344 days. Samples of 2 ml were taken at every time point and DNA was extracted using the Fast DNA Kit for Soil (MP Biomedicals, Solon, OH) according to the manufacturer's protocol with two 45-s beat beating steps using a Fastprep Instrument (MP Biomedicals). Afterwards, DNA was purified and concentrated using the DNA Clean and Concentrator kit (Zymo Research Corporation, Irvine, CA). DNA concentrations were determined with the Qubit 2.0 fluorometer (Thermo Fisher Scientific).

DNA was extracted from 0.5 to 1.0 g of sediment using a FastDNA Kit for Soil (MP Biomedical, USA) according to the manufacturer's instructions. DNA integrity was checked in a 1.0% agarose gel, and the concentration was measured using a ND-2000C spectrophotometer (NanoDrop, USA).
High-throughput sequencing of archaeal 16S rRNA genes was performed to reveal archaeal diversity and the community composition in the sediments. The V3 region of the archaeal 16S rRNA gene was PCR amplified with adapter-modified core primers, which contained unique 12 bp bar codes and the archaeal-specific primers A344F (5′-GGGGYGCASCAGGSG-3′) and A519R (5′-GGTDTTACCGCGGCKGCTG-3′). PCR was conducted using the following program: 94°C for 5 min; 25 cycles of 94°C for 50 s, 53°C for 50 s, and 72°C for 50 s; and a final extension at 72°C for 6 min [22 (link)]. The amplicons were gel purified and further purified with AMPure beads (Beckman Coulter, USA) and then pooled in equimolar proportions and sequenced on 318 chips with an Ion Torrent Personal Genome Machine (PGM) according to the manufacturer's instructions (Life Technologies, USA).

For DNA analyses, sediment samples (500 mg) listed in Supplementary Material Table S2 were used. The DNA extractions were performed using two isolation kits: NucleoSpin^® Soil (Macherey-Nagel, Düren, Germany) and a FastDNA™ Kit for Soil (MP Biomedicals, Santa Ana, CA, USA). To optimize the procedure, different modifications to manufacturer’s instructions were made (Supplementary Material Table S5). All extractions were performed in two variants: with and without a clean-up using Anty-Inhibitor Kit (A&A Biotechnology, Gdynia, Poland). The experimental variants and their results are presented in Supplementary Material Table S3. The presence of DNA was confirmed using a 1% agarose gel electrophoresis. The quality and quantity of extracted DNA was determined with SpectraMax^® i3 Platform (Molecular Devices LLC., Sunnyvale, CA, USA) equipped with SpectraDrop Micro-Volume Microplate.