Genomic DNA from each of the 18 selected E. coli isolates was extracted using GeneElute Bacterial Genomic DNA kit (Sigma-Aldrich, Oakville, ON, Canada) for WGS. The extracted DNA was stored in 1X TE buffer (pH 8.0), quantified by Invitrogen Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). The quality of DNA was visualized by 1% agarose gel electrophoresis and the DNA was then stored at −20 °C until further use.
Geneelute bacterial genomic dna kit
Manufactured by Merck Group
Sourced in United States, Sweden, Australia
The GeneElute Bacterial Genomic DNA Kit is a laboratory equipment product designed for the rapid and efficient purification of high-quality genomic DNA from a variety of bacterial species. It utilizes a silica-membrane-based technology to facilitate the isolation and concentration of DNA fragments from bacterial cell lysates.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
E z n a gel extraction kit, by Omega Bio-Tek (2 mentions)
Restriction endonuclease, by New England Biolabs (2 mentions)
Primestar gxl dna polymerase, by Takara Bio (2 mentions)
Nanodrop 1000c spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Qiagen (2 mentions)
Proteinase k, by New England Biolabs (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Pcr dig probe synthesis kit, by Roche (1 mentions)
Cdp star substrate, by Roche (1 mentions)
Hybond, by Cytiva (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Electroporator 2510, by Eppendorf (1 mentions)
24 protocols using geneelute bacterial genomic dna kit
1
Bacterial Genomic DNA Extraction
2
Genomic DNA Extraction from Saliva, Biofilm
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Genomic DNA was extracted from the saliva pellets, tooth biofilm samples, 3 mock communities, and a sample of ultra-pure water (negative control) with a Gene elute™ Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA) as described previously11 (link). Briefly, samples were (i) centrifuged for 5 min at 13,000 rpm, (ii) lysed in buffer with lysozyme and mutanolysin for 30 min at 37 °C, (iii) treated with RNase for 2 min at room temperature followed by Proteinase K for 10 min at 55 °C, (iv) mixed with ethanol and transferred to the binding column, and (v) washed and eluted in 100 µl of elution buffer. The DNA quality and quantity were evaluated using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) to meet the standard set by the sequencing facilities (OD 260/280 ratio ≥1.8). DNA was extracted from undivided samples, and after the DNA content had been determined, the volumes at concentrations requested by the sequencing facilities were subjected to further preparation.
3
Constructing Knockout Mutants of S. mutans
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To construct spaP and cnm knockout mutants in S. mutans wild type strain 49 (no. 23 in Fig. 2 , Fig. S2), a PCR overlapping strategy was used involving insertion of a selective marker (ermAD or add9) within the target gene. The DNA was purified using GeneElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Sweden), PCR amplifications were done with iProof High fidelity DNA polymerase (Bio-Rad, Sweden) and all primers (Table S2) were from Eurofins Genomics (Germany). Briefly, the 5′- and 3′-regions of the target gene were fused to the selective marker by end homology PCR and sub cloned into ZeroBlunt TOPO vector and transformed into a TOPO10 E. coli strain (Bio-Rad, Sweden). Correct generated disruption fragments were confirmed by sequencing (Eurofins Genomics, Germany). For construction of knockout mutants the cloned disruption fragments were amplified using T7 and Sp6 primers, transformed into isolates of S. mutans strain 49 by using a competence-stimulating peptide (SGSLSTFFRLFNRSFTQALGK) (Li et al., 2001 ). Correctly integrated fragments were confirmed by PCR and sequencing (Eurofins Genomics, Germany) (see also Fig. S5).
4
Bacterial Genomic DNA Extraction and Hybridization
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Genomic DNA was extracted using the GeneElute bacterial genomic DNA kit (Sigma-Aldrich), and 100 ng from each genome was spotted onto Hybond N+ membrane (GE Healthcare, Amersham), UV cross-linked, and hybridized with specific pESI backbone incP and rpoD digoxigenin (DIG)-labeled probes. The probes were labeled using the PCR DIG probe synthesis kit (Roche). The blots were hybridized overnight at 45°C in a DIG-Easy hybridization solution and washed twice with 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 mM sodium citrate)–0.1% SDS at room temperature, followed by a 15-min wash with 0.1× SSC–0.1% SDS at 68°C. The detection was done using anti-digoxigenin–alkaline phosphatase (AP), Fab fragments, and CDP-Star substrate (Roche).
5
Isolation and Genomic DNA Extraction of Tannerella forsythia
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The type strain of T. forsythia (ATCC 43037 = FDC 338) was obtained from ATCC (Manassas, VA, USA) and grown under anaerobic conditions in brain–heart infusion broth with supplements as described previously [12 (link)]. Bacterial DNA was extracted using the GeneElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Vienna, Austria) following the manufacturer’s protocol. The quality of the genomic DNA was checked on a 0.6% standard agarose gel stained with ethidium bromide, and using a NanoDrop ND-1000 spectrophotometer (ThermoFisher, Waltham, MA, USA). Quantification was performed using a Qubit 3.0. fluorometer together with a dsDNA BR assay kit (ThermoFisher, Waltham, MA, USA).
6
Bacterial DNA and RNA Extraction
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Genomic DNA from 1 ml of bacterial cultures was extracted with the GeneElute Bacterial Genomic DNA Kit (SIGMA), quantified using NanoDrop spectrophotometer (Nanodrop Technologies), and its integrity was evaluated by agarose gel electrophoresis. For RNA-seq experiments, total RNA was extracted and treated as previously described [40] (link). Total RNA extraction for qRT-PCR was performed using RNeasy minikit (QIAGEN) from 1 ml of BtCDC272 overnight cultures (ca. 16 hours growth, Figure S1 ). RNA samples were checked by agarose gel electrophoresis to assess the lack of degradation and then quantified with NanoDrop spectrophotometer. Genomic DNA degradation and reverse transcription were performed on 1 μg of total RNA using QuantiTect Reverse Transcription kit (QIAGEN).
7
Genomic DNA Extraction and Manipulation
8
Routine Molecular Cloning Procedures
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Routine molecular manipulations and cloning procedures were carried out as described in Sambrook et al. (1989 ). T4 DNA ligase and restriction enzymes were purchased from Fermentas (Burlington, Canada). AccuPrep Plasmid Mini Extraction Kit and AccuPrep PCR Purification Kit were used for plasmid and PCR product extraction and purification, respectively (Bioneer Corporation, Daejeon, Republic of Korea). DNA was extracted with the GeneElute bacterial genomic DNA Kit (Sigma‐Aldrich, St Louis, MO, USA). PCR primers were purchased from Sigma‐Aldrich and are listed in Table S7 . PCRs were performed with the Readymix Red Taq PCR reactive mix (Sigma‐Aldrich) or with the Phusion high‐fidelity DNA polymerase (Fermentas, Waltham, MA, USA) using an Eppendorf (Hamburg, Germany) thermal cycler. Sequencing of PCR fragments and constructs was performed at Hy Laboratories (Rehovot, Israel). Escherichia coli strains were transformed using an Eppendorf 2510 electroporator according to manufacturer's instructions. Plasmid mobilizations to A. citrulli and X. euvesicatoria strains were done by biparental mating as described (Bahar et al., 2009 ). Agrobacterium tumefaciens cells were transformed by the heat shock method (Zhou et al., 2009 ).
9
Whole Genome Sequencing of Lactobacillus casei N87
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DNA for genome sequencing was isolated from the stationary phase culture of L. casei N87 by using the GeneElute Bacterial Genomic DNA Kit (Sigma-Aldrich). The quality and quantity of DNA was assessed using both agarose gel electrophoresis and a NanoDrop® 1000c spectrophotometer (Thermo Scientific, Wilmington, DE). The whole genome sequencing (WGS) of L. casei N87 was performed using an Illumina HiSeq 1000 platform (Centre of Functional Genomics, Department of Science and Technology, University of Verona, Italy) and the WGS shotgun project was deposited at DDBJ/EMBL/GenBank with the accession no. LCUN00000000 [21 (link)]. Sequences encoding for pyruvate oxidase (pox), for the subunit I of cytochrome D ubiquinol oxidase (cydA), for the subunit II of cytochrome D ubiquinol oxidase (cydB), for the ABC transporters of cytochrome D ubiquinol oxidase (cydC and cydD), for the glyceraldehyde-3-phosphate dehydrogenase (gapdh), for the elongation factor Tu (tuf) and for the subunit B of DNA gyrase (gyrB) were used as template for primer design (Primer Express software 3.0, Applied Biosystems, Concord, Ontario, Canada; S1 Table ).
10
DNA Extraction from O. oeni Strains
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For DNA extraction, O. oeni strains AWRIB429, AWRIB787, and ATCC BAA-1163 were grown to an optical density at 600 nm (OD600) of 1 in MRS medium (Amyl Media) supplemented with 20% apple juice. Cells were pelleted by centrifugation (15 min, 3,000 × g) and washed in 1 ml of GTE buffer (50 mM glucose, 25 mM Tris pH 8, and 10 mM EDTA). For cell lysis, pellets were resuspended in 1 ml of GTE buffer containing 20 mg/ml lysozyme and incubated for 3 h at 37°C. One hundred microliters of 10% SDS was then added and mixed before incubation for 40 min at 37°C. Then, 2 μl of RNase A (40 μg/μl) (Qiagen, Australia) was added, vortexed, and incubated for 20 min at 37°C. Twenty microliters of proteinase K (20 μg/μL) (New England BioLabs, Australia) was then added, mixed, and incubated overnight at 37°C. Finally, DNA was isolated and purified using a GeneElute bacterial genomic DNA kit (Sigma, Australia) following the manufacturer’s instructions.
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