The DNA extraction was performed using the Nucleospin Plant II Midi kit (Macherey–Nagel GmbH & Co. KG, Düren, Germany) with minor modification in the initial step of cell lysis. Instead of the PL1/PL2 cell lysis buffers provided with the kit, manually prepared buffers consisting of 2% CTAB (Cetyl trimethylammonium bromide) (see Supplementary Table S11 ) and 2% PVP (Polyvinylpyrrolidone) (2 g in 100 mL distilled water) were used. 500 μl of 2% CTAB and 200 μl of 2% PVP were used for 1 g of sample, and if this was not sufficient for the samples to dissolve, CTAB and PVP were increased proportionally. All remaining steps were followed according to the manufacturer’s instructions to obtain high-quality DNA. DNA quantification of all the samples was performed using the Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA).
Nucleospin plant 2 midi kit
Manufactured by Macherey-Nagel
Sourced in Germany
The Nucleospin Plant II Midi kit is a laboratory product designed for the purification of genomic DNA from plant materials. The kit utilizes a silica membrane technology to efficiently capture and purify DNA from a variety of plant sources.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Purelink plant total dna purification kit, by Thermo Fisher Scientific (1 mentions)
Nanovue plus, by GE Healthcare (1 mentions)
5 protocols using nucleospin plant 2 midi kit
1
Modified DNA Extraction Using CTAB and PVP
2
Bacterial Genome Sequencing and Analysis
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Standard molecular biology techniques were carried out as previously described (Sambrook et al., 1989 ). Plasmid DNA was prepared with a Roche High Pure plasmid isolation kit (Merck KGaA, Darmstadt, Germany). DNA fragments were purified with a Wizard SV Gel and PCR Clean‐Up System (Promega, WI, USA). Oligonucleotide primers were supplied by FASMAC (Kanagawa, Japan) and their sequences are listed in Table S3 . All cloned inserts and DNA fragments were confirmed by DNA sequencing through an Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific, MA, USA). Strain MBES04 was grown aerobically overnight with shaking at 30°C in the basal medium. Cells were collected by centrifugation at 10,000 × g for 5 min at 4°C. Genomic DNA samples were purified using a NucleoSpin Plant II Midi kit (MACHEREY‐NAGEL GmbH & Co., Dueren, Germany). Genome sequencing was conducted using PacBio RS II and Illumina MiSeq and assembled using CLC De novo assembly v. 9. The updated complete genome sequences of strain MBES04 were deposited in GenBank/DDBJ/EMBL at accession numbers AP026899–AP026901. KEGG Automatic Annotation Server (KAAS) was used for the KEGG orthologous (KO) group assignment with the SBH (single‐directional best hit) method set to 45 as the threshold assignment score (Moriya et al., 2007 (link)).
3
Cultivation and DNA Extraction of Diverse Algae
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Cultures of Chromulina chionophila (CCAP 909/9), Pseudopedinella elastica (SAG B43.88), Synura petersenii (CCAC 0052), Phaeomonas parva (CCMP 2877), and Florenciella parvula (RCC 446) were obtained from their respective algal culture collections (CCAP: https://www.ccap.ac.uk/ ; SAG: http://www.uni-goettingen.de/en/culture±collection±of±algae±%28sag%29/184982.html ; CCAC: https://www.uni-due.de/biology/ccac/ ; CCMP: https://ncma.bigelow.org/ ; RCC: http://roscoff-culture-collection.org/ ). Algae were grown in the culture media recommended by the collections in aerated 1-l Erlenmeyer flasks at 15 °C and 20 µmol photons/m2/s in a 14:10 h L/D cycle. They were harvested by centrifugation and, after grinding in liquid nitrogen, total DNA was extracted using either the NucleoSpin Plant II Midi Kit (Macherey-Nagel, Düren, Germany) or a modified CTAB protocol (Rogers and Bendich 1985 (link); see Supplementary Material online ).
4
Fungal and Plant DNA Extraction Protocols
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Fungal genomic DNA was isolated using 100 mg of mycelium from A. flavus cultures using PureLink Plant Total DNA Purification kit (Invitrogen, Carlsbad, CA, USA). The purified DNA was evaluated in 0.8% (w/v) agarose gel followed by quantitative and qualitative determination using Qubit® Fluorometer 2.0 and spectrophotometer (GE Healthcare, New Jersey, USA), respectively, and stored at −20 °C until use.
The plant genomic DNA was extracted from 1 g leaf samples from 30‐day‐old transgenic and wild‐type (WT) peanut using a standard protocol (Dellaporta et al.,1983 ) and quantified using NanoVue Plus™ (GE Healthcare). For the estimation of fungal load in the host tissues, genomic DNA from healthy and infected peanut cotyledon samples was isolated using NucleoSpin plant II midi kit (Macherey‐Nagel, Duren, Germany) following the manufacturer's protocol.
The plant genomic DNA was extracted from 1 g leaf samples from 30‐day‐old transgenic and wild‐type (WT) peanut using a standard protocol (Dellaporta et al.,
5
Cooked Ham Microbiota Evaluation
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Diced cooked ham (except for one sample consisting of sliced cooked ham) were purchased in 2015 and 2016 from local supermarkets in Nantes, France, transported at cold temperature to the laboratory and reconditioned immediately. These samples, sold as ready-to-eat food were conditioned in packs ranging from 120 to 200 g under modified atmosphere or without any indication of any gas mixture used in the packs. At arrival in the laboratory (day 1), bags were opened and dices were immediately reconditioned as 25 g aliquots under air or vacuum packaging and incubated at 4°C. Total counts and LAB counts were enumerated at day 1, 7, 14, 21, and 28. When PCA mesophilic counts reached about 7 log10 CFU per gram of ham, bacterial communities were collected by mixing 25 g diced cooked ham in 75 ml peptone salt (AES, France) for 2 min in a stomacher (Masticator, IUL Instruments, England). The homogenate was filtered through the bag filter and centrifuged through a filter from Nucleospin Plant II Midi kit (Macherey Nagel, EURL, France) at 8000 × g during 10 min at room temperature. The bacterial pellet was resuspended in 30 ml peptone salt and aliquoted as 1 ml with glycerol 15% and stored at -80°C. In total, eight microbiota were recovered from ham dices either conditioned under air or vacuum.
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