Typically, 100 mg of liquid N2 batch-frozen leaf tissue was ground with a mortar and pestle, and genomic DNA was isolated using the GenElute Plant Genomic DNA Kit (Sigma-Aldrich), according to the manufacturer's protocol.
Genelute plant genomic dna kit
Manufactured by Merck Group
Sourced in Germany, Sao Tome and Principe, United States
The GenElute Plant Genomic DNA kit is a product from Merck Group designed for the isolation and purification of genomic DNA from plant tissues. It provides a simple and efficient method for extracting high-quality plant DNA suitable for various downstream applications.
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Lab products found in correlation
6 protocols using genelute plant genomic dna kit
1
Genomic DNA Extraction from Leaf Tissue
2
Fungal DNA Extraction and Gene Identification
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Fungal genomic DNA for PCR was extracted from mycelia of A. oryzae NSAR1 grown in DPY medium by using the GenElute plant genomic DNA kit (Sigma-Aldrich, Darmstadt, Germany). Using genomic DNA as the PCR template, a pair of specific 5′ and 3′ primers for gene identification were designed. PCR product purification was carried out by using the Wizard SV gel and PCR clean-up system (Promega, Madison, WI, USA). The PCR product was further identified by gene sequencing.
Total RNA was isolated from fresh 2-day-old cultured mycelia of A. oryzae NSAR1 grown in DPY medium by using the Quick-RNA™ Fungal/Bacterial Miniprep Kit according to the manufacturer′s protocol (Zymo Research, Irvine, CA, USA). Single-stranded cDNA was prepared from total RNA by using the High-Capacity RNA-to-cDNA™ Kit (ThermoFisher, Waltham, MA, USA). Using the cDNA as a template, the PCR reaction with specific 5′ and 3′ primers for gene identification was carried out. For the gene identification under the condition of adding propionate, 50 mM of sodium propionate was supplemented into the A. oryzae culture one day before total RNA isolation.
Total RNA was isolated from fresh 2-day-old cultured mycelia of A. oryzae NSAR1 grown in DPY medium by using the Quick-RNA™ Fungal/Bacterial Miniprep Kit according to the manufacturer′s protocol (Zymo Research, Irvine, CA, USA). Single-stranded cDNA was prepared from total RNA by using the High-Capacity RNA-to-cDNA™ Kit (ThermoFisher, Waltham, MA, USA). Using the cDNA as a template, the PCR reaction with specific 5′ and 3′ primers for gene identification was carried out. For the gene identification under the condition of adding propionate, 50 mM of sodium propionate was supplemented into the A. oryzae culture one day before total RNA isolation.
3
Plant Genomic DNA Extraction and Quantification
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Total genomic DNA of each accession was extracted from young leaves using the GenElute Plant Genomic DNA kit (Sigma Aldrich) according to manufacturer’s instructions20 . DNA quantity and quality were estimated from a 0.8% (w/v) agarose gel with electrophoresis at 80 V in UNTAN (40 mM Tris–Cl; 2 mM EDTA, pH adjusted to pH 7.4 with acetic acid) buffer. DNA was visualized with ethidium bromide staining under UV light. The concentration of the DNA samples was determined by using a UV spectrophotometer and measuring absorbance at A260 and A280. Samples were diluted to 20 ng/μl for SSR analysis.
4
Extraction of Plant Genomic DNA
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The plant materials consisted of 12 accessions from East Java, 44 from West Java, 16 from East Africa, 30 from Central Africa, 24 from Southern Africa and 44 from West Africa (Table S1) were planted in the climate-controlled glasshouse located at the Sutton Bonington Campus of University of Nottingham, UK. DNA was extracted from young leaflets using the GenElute Plant Genomic DNA kit (Sigma Aldrich) according to the manufacturer's instructions (Basu et al., 2007; Molosiwa, 2015) . The DNA quality and quantity were evaluated under UV light on 1% Tris-borate-EDTA (TBE) agarose gel stained with ethidium bromide.
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5
Sponge Microbiome DNA Extraction
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Sponge fragments (∼100 mg per sponge individual) were washed with autoclaved and 0.2 µm-filtered ambient water (AW) obtained from the studied marine aquaria system and stored at -20 • C. In 2015, water-associated bacteria were collected before sponge sampling from 1 L surrounding aquarium water of three locations of the tank on 0.22 µm Sterivex TM -GP filter units (Merck Millipore, Burlington, United States). Remaining water was fully removed from the cartridges and filter units were frozen in liquid nitrogen. Frozen plastic cartridges of the filter units were broken by mechanical treatment and filters subjected to DNA extraction. Total DNA was extracted from sponge samples and filters using the Gen Elute Plant Genomic DNA Kit (Sigma-Aldrich, St. Louis, United States). All samples were homogenized by bead-beating with 1 g of silica beads in the lysis solution of the kit using a Retsch mill (MM2; Retsch GmbH, Haan, Germany) for 1 min at maximum speed. Subsequent DNA extraction was performed according to the manufacturer's protocol using two elution steps with 100 µL elution solution each and stored at -20 • C.
6
Aflatoxin-Producing Fungal Isolates Identification
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The Aspergillus isolates were cultured in 0.5 ml of malt extract broth medium (30 g malt extract and 5 g peptone l -1 ) in Eppendorf tubes at 25°C for three days, after which the mycelium was transferred to a new Eppendorf tube. DNA was extracted from isolates using both octanol/isopropanol method as described by Paavanen-Huhtala et al. (1999) and GenElute™ Plant Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO, USA) as described by Yli-Mattila et al. (2008) . Quality of DNA extraction was confirmed using ITS1 and ITS4 (Table 2) primers amplifying ITS (the internal transcribed spacer) region of fungi DNA as described by Yli-Mattila et al. (2004) .
[XE "Multilocus genotyping"] Primers pairs ver-1/ver-2 and ordAF/ordAR (Table 2) which are specific for ver-1 and ordA genes in aflatoxin biosynthetic pathway respectively, were used for the detection of aflatoxin production by aflatoxigenic isolates as described by Färber et al. (1997) and Chang et al. (2005) (Table 2). MJ Research thermal cycler (PTC-200) was used for PCR amplification.
Table 2 Sequences of primer pairs used in PCR: ITS 1/ITS4 for testing the quality of DNA and ver-1/ver-2 and ordA-F/ordA-R for detecting ver-1 gene and ordA gene in aflatoxin-producing fungi respectively.
[XE "Multilocus genotyping"] Primers pairs ver-1/ver-2 and ordAF/ordAR (Table 2) which are specific for ver-1 and ordA genes in aflatoxin biosynthetic pathway respectively, were used for the detection of aflatoxin production by aflatoxigenic isolates as described by Färber et al. (1997) and Chang et al. (2005) (Table 2). MJ Research thermal cycler (PTC-200) was used for PCR amplification.
Table 2 Sequences of primer pairs used in PCR: ITS 1/ITS4 for testing the quality of DNA and ver-1/ver-2 and ordA-F/ordA-R for detecting ver-1 gene and ordA gene in aflatoxin-producing fungi respectively.
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