Genomic DNA was prepared using the QIAGEN® Genomic-tip 20/G kit (QIAGEN, Germany). Libraries for NGS were prepared using the Nextera XT DNA Library Preparation Kit (Illumina Inc., USA). Paired-end sequencing (2*300 bp) was performed with an Illumina MiSeq instrument (Illumina Inc., USA).
Genomic tip 20 g kit
Manufactured by Qiagen
Sourced in Germany, United States
The Genomic-tip 20/G kit is a product manufactured by Qiagen that is used for the isolation and purification of high-molecular-weight genomic DNA from a variety of sources, including cells, tissues, and microorganisms. The kit utilizes a gravity-flow anion-exchange resin-based column to capture and purify the DNA, which can then be used for downstream applications such as PCR, sequencing, or other molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (6 mentions)
Nextera xt dna library preparation kit, by Illumina (4 mentions)
Picogreen dsdna reagent, by Thermo Fisher Scientific (4 mentions)
Miseq instrument, by Illumina (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (2 mentions)
Genomic dna buffer set, by Qiagen (2 mentions)
Miseq platform, by Illumina (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Pacbio rs 2 system, by Pacific Biosciences (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Nutrient agar, by Merck Group (1 mentions)
Nucleobond hmw dna kit, by Macherey-Nagel (1 mentions)
Nextera xt preparation kit, by Illumina (1 mentions)
Nextera xt library prep kit, by Illumina (1 mentions)
57 protocols using genomic tip 20 g kit
1
Genomic DNA Extraction and NGS Sequencing
2
Whole Genome Sequencing of Bacterial Pathogens
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Six strains of F. tularensis subsp. holarctica were cultivated on cysteine heart agar (CHA, Becton Dickinson, BD Heidelberg, Germany) at 37 °C with 5% CO2 for 72 h. The DNA used for whole genome sequencing was prepared using the QIAGEN Genomic-tip 20/G Kit (Qiagen GmbH, Hilden, Germany). The DNA extraction was performed according to the instructions of the manufacturer for sample preparation and the lysis protocol for bacteria using 1 ml buffer B1 with 2 µl RNase A, 45 µl proteinase K, and 20 µl lysozyme.
Cultivation of 6 strains each of Ba. anthracis and Br. suis biovar 2 was performed at 37 °C on nutrient agar (Merck, Darmstadt, Germany) for 24 h and on nutrient agar with 7.5% calf blood for 48 h, respectively. High molecular weight DNA was extracted using the NucleoBond HMW DNA kit (MACHEREY–NAGEL, Düren, Germany).
Cultivation of 6 strains each of Ba. anthracis and Br. suis biovar 2 was performed at 37 °C on nutrient agar (Merck, Darmstadt, Germany) for 24 h and on nutrient agar with 7.5% calf blood for 48 h, respectively. High molecular weight DNA was extracted using the NucleoBond HMW DNA kit (MACHEREY–NAGEL, Düren, Germany).
3
Next-generation sequencing of bacterial strains
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To perform next-generation sequencing, the fifteen strains were grown overnight at 37°C in 3 ml of Luria-Bertani broth (Mast Diagnostica GmbH, Reinfeld, Germany). The genomic DNA was extracted and purified using the QIAGEN® Genomic-tip 20/G kit (QIAGEN, Germany) and the Genomic DNA Buffer Set (QIAGEN, Germany). The concentration of the DNA was determined using the Qubit dsDNA BR assay kit (Invitrogen, United States). DNA sequencing libraries were constructed using the Nextera XT Preparation Kit (Illumina Inc., San Diego, CA, United States) following the manufacturer’s instructions. Paired-end sequencing was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, United States) using a 300-cycle MiSeq reagent kit.
4
Bioinformatic Analysis of E. coli Strains
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The Qiagen Genomic-tip 20/G kit (Qiagen) was used to extract the total genomic DNA following the manufacturer’s recommendations. For Illumina sequencing by MiniSeq, a Nextera XT Library Prep Kit and a Nextera XT Index Kit was used to prepare the DNA library (Illumina, San Diego, CA, United States) according to the manufacturer’s instructions. For Nanopore sequencing by GridION, construction of the library was performed by the SQK-RBK004 Rapid Barcoding Kit (Oxford Nanopore Technologies, Oxford, United Kingdom). The library was loaded onto a FLO-MIN106 R9.4.1 flow cell and sequenced with the GridION device (Oxford Nanopore Technologies, Oxford, United Kingdom). A hybrid assembly of MiniSeq short reads and Nanopore long reads was achieved by Unicycler (Wick et al., 2017 (link)). The annotation was performed using DFAST.3 The complete genome sequences of the two E. coli strains were investigated at the Center for Genomic Epidemiology4 using ResFinder-4.1 (identity threshold for gene predictions was 90%), MLST 2.0, pMLST 2.0, VirulenceFinder-2.0 and PlasmidFinder-2. Genomic comparisons were performed using the BRIG tool5 and EasyFig tool.6 The BLAST program7 and ISfinder8 were used to analyze the plasmids.
5
Quantitative PCR for Pneumococcal Carriage
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Carriage density by qPCR was determined by partial amplification of the lytA gene. The primer and probe sequences were as follows: forward primer, 5′-ACGCAATCTAGCAGATGAAGCA-3′; reverse primer, 5′-TCGTGCGTTTTAATTCCAGCT-3′; probe, 5′-(FAM)-GCCGAAAACGCTTGATACAGGGAG-(BHQ1)-3′ [21 (link)]. The 20-μL PCR mix consisted of 1 × TaqMan Universal PCR Master Mix (Life Technologies, Bleiswijk, The Netherlands), 0.1 μM each primer, 0.1 μM probe, and 1 μL of the extracted DNA. Thermal cycling was performed in an ABI 7500 Fast Real-Time PCR System (Life Technologies) under the following cycling conditions: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. A standard curve of a ten-fold dilution series of genomic DNA extracted from S. pneumoniae (TIGR4, ATCC BAA-334) was used. The genomic DNA was extracted with the Qiagen Genomic-tip 20/G Kit (Qiagen), and quantified with a spectrophotometer (Nanodrop ND-1000; Thermo Fisher Scientific, Landsmeer, The Netherlands). The conversion from weight of pneumococcal DNA to number of S. pneumoniae DNA copies was based on the weight of one genome copy of TIGR4 calculated as the genome length in base pairs times the weight of a DNA base pair (650 Da). The lower limit of detection was set at 40 cycles. A NW was considered to be positive if both duplicates yielded a qPCR signal below 40 cycles.
6
Genomic DNA Extraction and Sequencing
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The QIAGEN® Genomic-tip 20/G kit (QIAGEN, Germany) was used to prepare genomic DNA. NGS libraries were prepared using the NextEra XT DNA Library Preparation Kit (Illumina Inc., USA). An Illumina MiSeq instrument (Illumina Inc., USA) was used for paired-end sequencing. Raw sequences from this study are available and were deposited in the European Nucleotide Archive (ENA) with bio project accession PRJEB56537 in the ENA bio project database: https://www.ebi.ac.uk/ena/browser/view/PRJEB56537 .
7
Cultivation and DNA Extraction of P. stutzeri
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P.
stutzeri strain ATCC 14405 (=CCUG 16156) is a gamma-proteobacterium
that was originally isolated from a marine environment, which has
served as a model organism for denitrification studies.18 (link) Recent phylogenomic analyses of the genus Pseudomonas suggested to split it into several genera,
including Stutzerimonas with Stutzerimonas stutzeri as type species.19 (link) Cell growth: P. stutzeri cells were essentially grown as described,20 (link) cells were harvested at an OD600 nm of 1.6, flash-frozen
in liquid nitrogen, and stored at −80 °C (further details
including description of aerobic and oxygen limited conditions inSupplemental Methods ). DNA extraction: Genomic
DNA was extracted and purified using a QIAGEN Genomic-tip 20/G kit
(Qiagen) according to the manufacturer’s instructions.
stutzeri strain ATCC 14405 (=CCUG 16156) is a gamma-proteobacterium
that was originally isolated from a marine environment, which has
served as a model organism for denitrification studies.18 (link) Recent phylogenomic analyses of the genus Pseudomonas suggested to split it into several genera,
including Stutzerimonas with Stutzerimonas stutzeri as type species.19 (link) Cell growth: P. stutzeri cells were essentially grown as described,20 (link) cells were harvested at an OD600 nm of 1.6, flash-frozen
in liquid nitrogen, and stored at −80 °C (further details
including description of aerobic and oxygen limited conditions in
DNA was extracted and purified using a QIAGEN Genomic-tip 20/G kit
(Qiagen) according to the manufacturer’s instructions.
8
Bacterial DNA Extraction and Purification
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A loopful from each fresh culture was suspended in 1 mL sterilized phosphate buffer saline (PBS) and heat inactivated at 96°C for 30 min. Genomic DNA was extracted and purified from bacterial cultures using QIAGEN Genomic-tip 20/G Kit (Qiagen GmbH, Hilden, Germany) according to the instructions of the manufacturer with a prior in-house modification step, i.e., adding of a lysis mixture (10–20 μL lysostaphin, 20 μL lysozyme, 2 μL ribonuclease A (2 μL of 10 mg/mL) and 45 μL proteinase K) followed by an incubation at 37°C for 2 h with slight shaking.
The concentration and quality of eluted DNA was determined photometrically using a Colibri spectrophotometer (Titertek, Berthold Technologies GmbH & Co. KG, Germany) and additionally measured using a Qubit 3 fluorometer (Fisher Scientific GmbH, Dreieich, Germany). The prepared DNA was preserved at −20°C for further investigations.
The concentration and quality of eluted DNA was determined photometrically using a Colibri spectrophotometer (Titertek, Berthold Technologies GmbH & Co. KG, Germany) and additionally measured using a Qubit 3 fluorometer (Fisher Scientific GmbH, Dreieich, Germany). The prepared DNA was preserved at −20°C for further investigations.
9
Bacterial DNA Extraction and Quantification
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Genomic DNA was extracted and purified from a 48 h bacterial culture on Mueller-Hinton blood agar plates (Oxoid Deutschland GmbH, Wesel, Germany) using QIAGEN® Genomic-tip 20/G Kit (QIAGEN®, Hilden, Germany) according to the manufacturer's instructions. The DNA was eluted in 200 μl elution buffer. DNA was quantified spectrophotometrically using a Nanodrop® ND-1000 (Fisher Scientific GmbH, Schwerte, Germany). The quality of the DNA was determined using the Qubit dsDNA BR Assay Kit (Invitrogen, Carlsbad CA, USA).
10
10x Chromium Genome Sequencing
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High molecular weight (HMW; >50 Kb) genomic DNA (gDNA) was extracted with the QIAGEN Genomic-tip 20/G kit (10223, QIAGEN) from nucleated erythrocytes of an adult female leucistic individual. The resulting gDNA was used for a 10x Chromium library preparation (Novogene Co., Ltd.) with the Chromium Genome HT Library Kit and Gel Bead Kit v2 (Zheng et al., 2016) (link). We sequenced the resulting barcoded fragments with a HiSeq X instrument, obtaining 522 million 150 bp pairedend reads.
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