Rhizoctonia solani (GenBank Accession No.) was isolated from infected tomato plants [37 ]. Fungal spore cultures of the pathogen were purified and kept on potato dextrose agar (PDA) media and stored at 4 °C until further bioassay. Tomato (Solanumly copersicum L.) seeds of (Super strain B) variety were obtained from the Ministry of Agriculture, Egypt. Thirty sterilized conical flasks (250 mL) containing PD broth were seeded with ten selected fungal isolates (three repeats for each isolate) and incubated at 28 ± 2 °C. After 5 to 7 days of fungal inoculation on PDA media, approximately 100 mg of mycelial biomass were harvested [8 (link)]. The genomic DNA of each isolate was extracted using Biospin Fungus Genomic DNA Extraction Kit (Bioer, Hangzhou, China), following the manufacturer’s protocol. Purified DNAs were transferred into new tubes and stored at −20 °C until processing. The ITS region in the rDNA repeat of the 28S gene was amplified using primer (Table S1 ) [38 (link)]. PCR amplification was carried out in a thermocycler ABI Gene Amp 9700 (Applied Biosystems, Waltham, MA, USA) accordingly. The obtained PCR product of ITS1 and ITS4 regions were sequenced using ABI PRISMTM 3100 DNA sequencer (Applied Biosystems) and Big Dye terminator sequencing kit (Version 3.1, Applied Biosystems, USA).
Biospin fungus genomic dna extraction kit
Manufactured by Bioer
Sourced in China
The Biospin Fungus Genomic DNA Extraction Kit is a laboratory equipment designed for the isolation and purification of genomic DNA from fungal samples. It utilizes a quick and efficient spin-column-based method to extract high-quality DNA suitable for downstream molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 3100 dna sequencer, by Thermo Fisher Scientific (1 mentions)
Abi geneamp 9700, by Thermo Fisher Scientific (1 mentions)
Seqman pro software, by DNASTAR (1 mentions)
Primestar max premix, by Takara Bio (1 mentions)
S1000 thermal cycler, by Bio-Rad (1 mentions)
16 protocols using biospin fungus genomic dna extraction kit
1
Molecular Identification of Rhizoctonia solani
2
Genomic DNA Extraction and Bioinformatic Analysis of Crac in Trichoderma rosea
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A Biospin fungus genomic DNA extraction kit (Bioer Technology Co., Ltd., Hangzhou, China) was used to extract the genomic DNA of C. rosea 67-1. Full-length crac was then amplified using primer pairs AC-F and AC-R through PCR (Table 1 ). The PCR products were detected by electrophoresis and sequenced at Sangon Biotech Co., Ltd. (Shanghai, China) Sequence information of crac was submitted to GenBank to obtain accession numbers.
The ExPASy program was used to predict the molecular weight and isoelectric point of crac [22 ]. SignalP 6.0 and TMPRED software were used to detect the signal peptide and transmembrane regions of crac, respectively [23 (link),24 ]. MEGA 6.0 software was used to conduct phylogenetic analysis of crac, with 1000 replications under the neighbor-joining method [25 (link),26 (link)].
The ExPASy program was used to predict the molecular weight and isoelectric point of crac [22 ]. SignalP 6.0 and TMPRED software were used to detect the signal peptide and transmembrane regions of crac, respectively [23 (link),24 ]. MEGA 6.0 software was used to conduct phylogenetic analysis of crac, with 1000 replications under the neighbor-joining method [25 (link),26 (link)].
3
Fungal DNA Extraction and Sequencing Protocol
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Fungi were grown on PDA for 20–30 days at 25°C. A Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Hangzhou, China) was used to extract total genomic DNA from fresh mycelia according to the manufacturer’s instructions. DNA amplification was performed by polymerase chain reaction (PCR). LSU, SSU, ITS and RPB2 gene regions were amplified using the primer pairs LR0R/LR5, NS1/NS4, ITS5/ITS4 and RPB2-5F/RPB2-7cR, respectively (Vilgalys and Hester, 1990 (link); White et al., 1990 (link); Rehner and Samuels, 1994 (link); Liu et al., 1999 (link)). The amplifications were carried out in a 25 μL reaction volume containing 9.5 μL ddH2O, 12.5 μL 2 × PCR Master Mix, 1 μL DNA template, 1 μL each primer (10 μM). The PCR thermal cycles for the amplification of the gene regions followed the methods in Jeewon et al. (2004) ; Réblová et al. (2011) (link), and Su et al. (2015) (link). PCR products were checked on 1% agarose electrophoresis gels stained with Gel Red. The sequencing reactions were carried out by Shanghai Sangon Biological Engineering Technology and Services Co., Shanghai, China.
4
Fungal Genome Extraction and Sequencing
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Fungal mycelia were scraped from the colonies on PDA. The Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Hangzhou, China) was used to extract total genomic DNA. The polymerase chain reaction (PCR) technique was utilized for the amplification of target DNA fragments. The primer pairs LR0R/LR5, NS1/NS4, ITS5/ITS4 and fRPB2-5F/fRPB2-7cR were used to amplify LSU, SSU, ITS and rpb2 [20 (link),21 (link),22 (link)]. The amplifications were carried out as detailed in Dong et al. [23 (link)]. The PCR thermal cycle program for the amplification of LSU, SSU and ITS was provided as initially 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 50 s, elongation at 72 °C for 90 s and a final extension at 72 °C for 10 min. The annealing was adjusted to 52 °C for rpb2. PCR products were checked on 1% agarose electrophoresis gels stained with Gel Red. The sequencing reactions were carried out by Shanghai Sangon Biological Engineering Technology and Services Co., Shanghai, China.
5
Fungal Biomass DNA Extraction Protocol
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Then, 250-mL sterilized conical flasks containing PD broth were inoculated with each fungal isolate (three replicates/isolate). All flasks were incubated at 28 ± 2 °C for a week and about 100 mg of mycelial biomass was gathered to be processed for DNA extraction. The total DNA of each isolate was purified using the Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co. Ltd., Hangzhou, China). The manufacturer’s protocol was followed. Purified DNAs were stored in new tubes at –20 °C until further processing. The concentration, purity and integrity of DNA were assessed as previously described [43 ].
6
Genomic DNA Extraction and ITS Sequencing of New Fungus Species
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Genomic DNA was extracted from three dried specimens of the new species using a Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Ltd., Hangzhou, P. R. China). The ITS gene region of the new species was amplified. Amplification was performed in 25 µl volumes containing 1.0 µl template DNA, 9.5 µl double distilled water, 1.0 µl of each primer (ITS1/ITS4) [49 ] and 12.5 µl of 2× power Taq PCR Master Mix (A premix and ready to use solution, including 0.1 Units/µl Taq DNA Polymerase, 500 µM dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mM Tris–HCl (pH8.3), 100 mM KCl, 3 mM MgCl2, stabilizer and enhancer. The reaction was carried out with 35 cycles under the following conditions: denaturation (95 °C, 30 s), annealing (52 °C, 30 s), extension (72 °C, 1 min), and final extension (72 °C, 10 min). The primers used for sequencing the whole ITS region were the same as those used in White et al. [49 ]. Amplified products were confirmed with 1% agarose gel electrophoresis stained with ethidium bromide. The amplified PCR fragments were sent to a commercial sequencing provider (Beijing Bai Mai Hui Kang Biological Engineering Technology Co., Beijing, P.R. China). The newly generated sequence data of the new species were deposited in GenBank.
7
Fungal Genomic DNA Extraction and Sequencing
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Genomic DNA extraction was performed using the Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Ltd., Hangzhou, China) following the manufacturer’s instructions. PCR amplification was carried out targeting the internal transcribed spacers (ITS) region, β-tubulin gene, and calmodulin gene using the primers listed in Table S1, and the length of the PCR products were 621 bp, 436 bp, and 751 bp, respectively. The PCR products were then sent to BGI Genomics for sequencing. The sequences were analysed against the CBS database (https://wi.knaw.nl/page/Pairwise_alignment ).
8
Fungal DNA Extraction from Respiratory Samples
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DNA was extracted from sputum and BALF specimens using a commercial kit (Biospin Fungus Genomic DNA Extraction Kit, Bioer, Japan) according to the manufacturer’s instructions. After washing the spin column, 100 μL DNA was collected in a 1.5 ml tube by centrifugation. Extracted DNA was then directly used for PCR or stored at -20°C for later experiments.
9
Aspergillus niger Isolation and DNA Extraction
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Example 3
The Aspergillus niger strain NN053297 was isolated from hot spring soil samples collected from Yunnan province in 2010.
Aspergillus niger strain NN053297 was inoculated on PDA plate and incubated for 37 C for 4 days. Mycelia were collected and frozen in liquid nitrogen in a sterilized mortar and grounded with pestle to fine powders. Then the genomic DNA was extracted with Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co. Ltd., Hangzhou, China) following the manufacturer's instruction.
10
Fungal DNA Extraction and Sequencing
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DNA was isolated from samples taken from the dried specimens, using the Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology, Hangzhou, China), following the manual’s procedure. The DNA loci amplified by PCR were the ITS region (including ITS1, 5.8S, ITS2) with the primers ITS1-F and ITS4 (White et al. 1990 (link); Gardes and Bruns 1993 (link)), and nrLSU, with the primers LR0R and LR5 (Vilgalys and Hester 1990 (link); White et al. 1990 (link)). PCR products were purified and sequenced in both directions, using the PCR primers, by Sangon Biological Engineering Technology and Services (Shanghai, China). The quality of each generated sequence read was checked using Bioedit Sequence Alignment Editor version 7.0.9.0 (Hall 1999 ) and sequence reads were assembled using SEQMan Pro software (DNA Star, Madison, USA).
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