For each monosporic strain, 5 mycelium plugs were collected from the margin of actively growing colonies on PDA and transferred to a sterilized cellophane disk overlaid on PDA 90 mm plates. After incubation at 25 °C for 2–3 days, the mycelia were collected into 2 mL tubes, frozen, and lyophilized. The DNA extraction was carried out on 15 mg of powdered lyophilized mycelium by using a Wizard Magnetic DNA Purification System for Food kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol. The genomic DNA quantity and integrity were checked on 0.8% agarose gel in 1X Tris-Acetate-EDTA (TAE) using a standard 1 kb DNA Ladder (ThermoFisher Scientific, Carlsbad, CA, USA) and a Nanodrop spectrophotometer (ThermoFisher Scientific).
Wizard magnetic dna purification system for food kit
Manufactured by Promega
Sourced in United States
The Wizard® Magnetic DNA Purification System for Food kit is a laboratory equipment product designed for the extraction and purification of DNA from food samples. The kit utilizes magnetic bead technology to efficiently capture and isolate DNA, allowing for further downstream processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sephadex g 50, by Merck Group (2 mentions)
Bigdye terminator v3.1 cycle sequencing ready reaction kit, by Merck Group (2 mentions)
3730 dna analyzer, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
1 kb dna ladder, by Thermo Fisher Scientific (1 mentions)
5 protocols using wizard magnetic dna purification system for food kit
1
Fungal Genomic DNA Extraction Protocol
2
Molecular Phylogeny of Fungal Strains
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DNA was extracted and purified from fresh mycelium using the "Wizard® Magnetic DNA Purification System for Food" kit (Promega) according to the manufacturer's protocol.
Phylogenetic relationships were investigated by amplifying and sequencing the housekeeping genes translation elongation factor 1-α (TEF), calmodulin (CAL) and βtubulin (TUB). Fragments of TEF gene were amplified using the primer pair EF1 and EF2 2000) and the TUB gene were amplified using the primers Bt2a and Bt2b (Glass & Donaldson 1995) . Before sequencing, PCR products were purified with the enzymatic mixture EXO/FastAP (Exonuclease I, FastAP thermosensitive alkaline phosphatase, Thermo Scientific). Sequence reactions were performed using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit for both strands, after which they were purified by gel filtration through Sephadex G-50 (5%) (Sigma Aldrich), before they were analyzed on the 3730 DNA Analyzer (Applied Biosystems).
Phylogenetic relationships were investigated by amplifying and sequencing the housekeeping genes translation elongation factor 1-α (TEF), calmodulin (CAL) and βtubulin (TUB). Fragments of TEF gene were amplified using the primer pair EF1 and EF2 2000) and the TUB gene were amplified using the primers Bt2a and Bt2b (Glass & Donaldson 1995) . Before sequencing, PCR products were purified with the enzymatic mixture EXO/FastAP (Exonuclease I, FastAP thermosensitive alkaline phosphatase, Thermo Scientific). Sequence reactions were performed using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit for both strands, after which they were purified by gel filtration through Sephadex G-50 (5%) (Sigma Aldrich), before they were analyzed on the 3730 DNA Analyzer (Applied Biosystems).
3
Molecular Characterization of Fusarium and Alternaria Strains
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Seventy-five (26 from Ardebil and 49 from Kerman) Fusarium strains (Table 1 ) and 83 Alternaria strains (34 from Ardebil and 49 from Kerman) (Table 2 ) were selected as representative of the Fusarium and Alternaria populations from different parts of potato plants, viz. leaves, stems, tubers, and roots. Selection of the strains was based on morphological features, sampling location (geographical area and farm), and potato plant tissue type.
For genomic DNA extraction and molecular analyses, the cryopreserved strains at ISPA-CNR were refreshed on PDA and then cultured on cellophane disks overlaid on PDA Petri dishes. After 3 days of growth at 25 °C, mycelia were scraped, transferred to 2 ml microtubes, frozen and lyophilized. Ten to fifteen mg of powdered lyophilized mycelium were used for DNA extraction by using the “Wizard Magnetic DNA Purification System for food” kit (Promega Corporation, Madison, WI), based on the manufacturer's protocol. The quantity and quality of extracted DNA were examined with Thermo-Scientific Nanodrop (LabX, Midland, ON, Canada), and by 0.8 % agarose gel electrophoresis, in comparison with a standard DNA (1 kb DNA Ladder, Fermentas GmbH).
For genomic DNA extraction and molecular analyses, the cryopreserved strains at ISPA-CNR were refreshed on PDA and then cultured on cellophane disks overlaid on PDA Petri dishes. After 3 days of growth at 25 °C, mycelia were scraped, transferred to 2 ml microtubes, frozen and lyophilized. Ten to fifteen mg of powdered lyophilized mycelium were used for DNA extraction by using the “Wizard Magnetic DNA Purification System for food” kit (Promega Corporation, Madison, WI), based on the manufacturer's protocol. The quantity and quality of extracted DNA were examined with Thermo-Scientific Nanodrop (LabX, Midland, ON, Canada), and by 0.8 % agarose gel electrophoresis, in comparison with a standard DNA (1 kb DNA Ladder, Fermentas GmbH).
4
Molecular Phylogeny of Fungal Strains
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DNA was extracted and purified from fresh mycelium using the "Wizard® Magnetic DNA Purification System for Food" kit (Promega) according to the manufacturer's protocol.
Phylogenetic relationships were investigated by amplifying and sequencing the housekeeping genes translation elongation factor 1-α (TEF), calmodulin (CAL) and βtubulin (TUB). Fragments of TEF gene were amplified using the primer pair EF1 and EF2 2000) and the TUB gene were amplified using the primers Bt2a and Bt2b (Glass & Donaldson 1995) . Before sequencing, PCR products were purified with the enzymatic mixture EXO/FastAP (Exonuclease I, FastAP thermosensitive alkaline phosphatase, Thermo Scientific). Sequence reactions were performed using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit for both strands, after which they were purified by gel filtration through Sephadex G-50 (5%) (Sigma Aldrich), before they were analyzed on the 3730 DNA Analyzer (Applied Biosystems).
Phylogenetic relationships were investigated by amplifying and sequencing the housekeeping genes translation elongation factor 1-α (TEF), calmodulin (CAL) and βtubulin (TUB). Fragments of TEF gene were amplified using the primer pair EF1 and EF2 2000) and the TUB gene were amplified using the primers Bt2a and Bt2b (Glass & Donaldson 1995) . Before sequencing, PCR products were purified with the enzymatic mixture EXO/FastAP (Exonuclease I, FastAP thermosensitive alkaline phosphatase, Thermo Scientific). Sequence reactions were performed using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit for both strands, after which they were purified by gel filtration through Sephadex G-50 (5%) (Sigma Aldrich), before they were analyzed on the 3730 DNA Analyzer (Applied Biosystems).
5
Fungal DNA Extraction Protocol
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Fungal isolates were grown in Wickerham medium (40 g of glucose, 5 g of peptone, 3 g of yeast extract, 3 g of malt extract per liter) on a rotary shaker at 120 rpm for 48 h in the dark. Fresh mycelia were collected by vacuum filtration through No. 4 Whatman filter paper (Whatman Biosystems Ltd., Maidstone, UK), then frozen and lyophilized. Total genomic DNA was extracted using the "Wizard® Magnetic DNA Purification System for Food" kit (Promega, USA) according to the manufacturer's protocol, using 10 mg of dried mycelium.
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