D. coronatus were washed off the agar plates and collected on 10 μm-mesh nylon filters. The nematodes were transferred to a 1-l flask containing 100 ml of distilled water and incubated for 2 h to allow digestion of remaining food bacteria. Nematodes were collected by filtration, aliquoted ~200 mg into 2.2 ml tubes and stored at -80 °C. 200–400 mg of packed worms were ground in a mortar in liquid nitrogen and used for a single DNA/RNA preparation. Genomic DNA was purified with the Genomic-tip 500/G Kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA was purified by RNAgents Total RNA Isolation System (Promega, Fitchburg, WI, USA) and polyadenylated RNA was purified with a mRNA Purification Kit (GE Healthcare Life Sciences, Buckinghamshire, UK) using an Oligo(dT)-cellulose column.
Genomic tip 500 g kit
Manufactured by Qiagen
Sourced in Germany
The Genomic-tip 500/G kit is a laboratory product designed for the purification of high-molecular-weight genomic DNA from a variety of sample types, including cells, tissues, and microorganisms. It utilizes a gravity-flow anion-exchange resin to selectively bind and purify DNA, allowing for efficient and reliable extraction.
Automatically generated - may contain errors
Lab products found in correlation
Rnagents total rna isolation system, by Promega (1 mentions)
Mrna purification kit, by GE Healthcare (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Bhi broth, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Rsii sequencer, by Pacific Biosciences (1 mentions)
Smrt analysis v2, by Pacific Biosciences (1 mentions)
Luria broth, by BD (1 mentions)
Bluepippin, by Sage Science (1 mentions)
Hiseq instrument, by Illumina (1 mentions)
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
11 protocols using genomic tip 500 g kit
1
Nematode DNA and RNA Extraction
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D. coronatus were washed off the agar plates and collected on 10 μm-mesh nylon filters. The nematodes were transferred to a 1-l flask containing 100 ml of distilled water and incubated for 2 h to allow digestion of remaining food bacteria. Nematodes were collected by filtration, aliquoted ~200 mg into 2.2 ml tubes and stored at -80 °C. 200–400 mg of packed worms were ground in a mortar in liquid nitrogen and used for a single DNA/RNA preparation. Genomic DNA was purified with the Genomic-tip 500/G Kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA was purified by RNAgents Total RNA Isolation System (Promega, Fitchburg, WI, USA) and polyadenylated RNA was purified with a mRNA Purification Kit (GE Healthcare Life Sciences, Buckinghamshire, UK) using an Oligo(dT)-cellulose column.
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D. coronatus were washed off the agar plates and collected on 10 μm-mesh nylon filters. The nematodes were transferred to a 1-l flask containing 100 ml of distilled water and incubated for 2 h to allow digestion of remaining food bacteria. Nematodes were collected by filtration, aliquoted ~200 mg into 2.2 ml tubes and stored at -80 °C. 200–400 mg of packed worms were ground in a mortar in liquid nitrogen and used for a single DNA/RNA preparation. Genomic DNA was purified with the Genomic-tip 500/G Kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA was purified by RNAgents Total RNA Isolation System (Promega, Fitchburg, WI, USA) and polyadenylated RNA was purified with a mRNA Purification Kit (GE Healthcare Life Sciences, Buckinghamshire, UK) using an Oligo(dT)-cellulose column.
2
High-molecular-weight DNA Extraction and Sequencing from Candida krusei
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High molecular weight genomic DNA was isolated from C. krusei strain 81-B-5 (Scherer and Stevens 1987 (link); Beckerman et al. 2001 (link)) using a QIAGEN Genomic-tip 500/G kit (catalog # 10262). DNA was adapted using the SMRTbell Template Prep Kit, and sequenced using PacBio Technology (P6-C4 chemistry). A total of three SMRTcells were run, generating a total of 266,621 subreads with mean read length 5758, with a total of 1,535,304,314 bases (∼140× coverage). DNA was also adapted for Illumina sequencing, and a total of 16,953,446 paired 101b reads was generated on a HiSeq 2500.
3
Bacterial Genomic DNA Extraction and Evaluation
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Bacterial genomic DNA (gDNA) extraction was performed using 1 ml overnight culture from a single colony grown in Brain Heart Infusion (BHI) broth (Oxoid, UK). DNA was extracted using a Qiagen GenomicTip 500/G kit (Qiagen, UK) following the manufacturer’s instructions. Pulsed Field Gel Electrophoresis was performed to assess gDNA degradation, with 100 ul of each sample separated on a 1% Agarose gel (Manufacturer info) for 18 h at 6 V. Gels were stained with 10 ul of ethidium bromide and visualised using UV G Box, Syngene. Sample gDNA quantity and quality was measured using by Qubit assay on a Qubit fluorometer (Life Technologies, USA) according to the manufacturer’s instructions, and by measuring absorbance at 260 nm and 280 nm using a Nanodrop Spectrophotometer (Thermo Scientific, UK).
4
Long-Read Sequencing of ETEC Isolates
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DNA for Pacific Biosciences (PacBio) sequencing was prepared from ETEC isolates grown in LB medium to an optical density at 600 nm (OD600) of 0.3. DNA was extracted by the Qiagen Genomic-tip 500/G kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). For each sample, one DNA aliquot was sheared into 10-kbp fragments using a Genemachines HydroShear instrument (Digilab, Marlborough, MA, USA) and a second aliquot was sheared into 2-kb fragments using a Covaris instrument (Covaris, Woburn, MA). SMRTbell templates were constructed according to the manufacturer’s instructions (Pacific Biosciences, Menlo Park, CA, USA). Each library was sequenced on 1 SMRT cell on a Pacific Biosciences RSII sequencer according to the manufacturer’s instructions with 4-h movie time.
5
Whole Genome Sequencing of mcr-1 E. coli
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Whole Genome Sequence analysis of mcr-1 containing isolates was performed to further characterize the E. coli strain including the plasmid carrying the mcr-1 gene and other genes associated with antimicrobial resistance [6 (link)]. The genome sequence of the mcr-1 containing isolate was determined using the Pacific Biosciences RSII system from DNA prepared by the Qiagen Genomic Tip 500/G kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. De novo assembly was performed using SMRT®Analysis v2.3.0 (PacBio's bioinformatics software suite) with expected genome size of 5 Mbp and coverage of 30. The assembled sequence was analysed using Geneious software V8.0.5 (Biomatters, Auckland, New Zealand) and the online tools Resfinder, MLST, SeroTypeFinder and Plasmidfinder (http://genomicepidemiology.org/ ). The plasmid sequence was analysed in DNA plotter to generate a circular DNA map.
6
Genome Sequencing of Diverse Bacterial Strains
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Twenty-four phylogenetically diverse strains were grown individually in 10 mL of Luria broth (Becton Dickinson) overnight at 37 °C in a shaking incubator. After overnight growth, 250 mL of inoculum was transferred to a fresh 10 mL of LB where it was incubated for 3 hours under the same incubation conditions. DNA from the culture was extracted via the QIAGEN Genomic tip 500/G kit via manufacture instructions. Ten micrograms of total DNA were sheared using the Covaris G-tubes spun at 4200RPM then 4100RPM. Libraries were constructed using the PacBio SMRTbell Express Template Preparation Kit 2.0 (Pacific Biosciences). Non-competing barcoded samples were pooled in groups of 4 to 5 and sized selected on a Blue Pippin (Sage Science) High Pass DNA Gel Cassette using a 15 kb protocol. Final library pools were quantified and run on the PacBio Sequel II. In addition to the 24 strains sequenced here, 12 strains from a previous study underwent the same procedure.
7
Sequencing and Assembly of Bp Isolates
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Live bacteria were grown in a BioSafety Level 3 facility in DSO National Laboratories. Genomic DNA was extracted using the Qiagen Genomic Tip 500/G kit (Qiagen). Unique index-tagged libraries for each sample were created, and up to 33 separate libraries were sequenced per lane on an Illumina HiSeq instrument with 100 base paired-end reads. Libraries were constructed using an amplification-free method (Kozarewa et al. 2009 (link)). Raw Illumina data were split to generate paired-end reads, and assembled using a de novo genome-assembly program, Velvet v0.7.03 (Zerbino and Birney 2008 (link)), to generate a multicontig draft genome for each Bp isolate.
8
Genomic DNA Extraction and Sequencing
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Genomic DNA for long-read sequencing was extracted from GD02 using the Genomic-tip 500/G kit (Qiagen) according to the manufacturer’s protocol, with modifications. Oxford Nanopore sequencing was performed by the Microbial Genome Sequencing Center. Long and short reads were assembled with Unicycler as a short-read first-assembly pipeline (36 (link)). The genome was annotated with the NCBI Prokaryotic Genome Annotation Pipeline.
9
Shewanella algae Genomic DNA Extraction
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Shewanella algae strains (Table S1 ) were grown in LB medium at 37°C prior to DNA isolation with (i) the GenElute bacterial genomic DNA kit (Merck) for samples submitted for WGS with Illumina MiSeq or (ii) with the Genomic-tip 500/G kit (Qiagen) for samples submitted for Oxford Nanopore or PacBio sequencing, as detailed below.
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10
High-molecular-weight DNA Extraction from Streptomyces
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High molecular weight genomic DNA (20–160 kb) was extracted from four Streptomyces strains (DF, SFW, QF2, and JS) with the QIAGEN-Genomic-tip 500/G kit (10262) according to the manufacturer’s protocol for bacteria.
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