Fungal control DNA
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Transcript of Fungal control DNA
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The pathogen strain identification The pathogen strain identification based on toxin biosynthetic genes based on toxin biosynthetic genes in the Romanian wheat genotypesin the Romanian wheat genotypes
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Fungal control DNA
DNA extraction: FastDNA SPIN Kit for Soil Quiagen
Permits a rapid isolation of PCR-ready genomic DNA from fungal samples.
Culture media:
Potato Extract Glucose Agar
Cereal samples preparation
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DNA extraction and purification method - CTAB method
Quantification of the extracted DNA by spectrophotometric method
Thermo Scientific NanoDrop 8000
100 mg ground material was pre-treated with Celulace (Promega) for 2 hours at 65°C then mixed with 300 μl water and then 700 μl CTAB buffer was added together with 20 μl RNase solution (10 mg/ml) before incubation at 65 °C for 30 min. And then 10 μl proteinase K solution (20 mg/ml) was added before an incubation at 65 °C for 30 min.
samples were centrifuged at 12,000 ×g for 10 min and the supernatant was transferred to a tube with 500 μl chloroform, vortexed and centrifuged at 12,000 ×g for 15 min.
the upper layer was transferred to a new tube and 2 volumes of CTAB precipitation solution were added and the samples were incubated at RT for 60 min before centrifugation at 12,000 ×g for 5 min.
the pellet was dissolved in 350 μl 1.2 M NaCl and 350 μl chloroform was added before vortexing and centrifugation at 12,000 ×g for 10 min.
the upper layer was precipitated with 0.6 volumes of isopropanol and incubated at RT for 20 min and centrifuged at 12,000 ×g for 10 min.
the pellet was washed in 70% ethanol, vacuum dried and resuspended in 100 μl MQ water.
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Amplification was performed in a Corbett RESEARCH Thermal Cycler, folowing the indications from literature.
Amplicons were analyzed by electrophoresis on 2% agarose gel (Promega, USA) and visualized in Ethidium Bromide (0.4 ng/ml) presence.
PCR reagents: Go Taq Green Master Mix PCR kit from Promega 2X. 20 pmol of each primer. different concentrations of DNA template. in a final volume – 25 µl.
PCR analyses
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Romanian wheat samples
DON 7 biosynthetic gene
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DON 13 biosynthetic gene
Romanian wheat samples
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NIV 13 biosynthetic gene
Romanian wheat samples
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NIV 13 biosynthetic gene
Romanian wheat samples
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Fusarium graminearum
Fusarium culmorum
Corn samples
15 artificially infected samples
14 GM corn samples
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