The pathogen strain identification based on toxin biosynthetic

The pathogen strain identification based on toxin biosynthetic genes in the Romanian wheat genotypes

Fungal control DNA Culture media: Potato Extract Glucose Agar DNA extraction: Fast. DNA SPIN Kit for Soil Quiagen Permits a rapid isolation of PCR-ready genomic DNA from fungal samples. Cereal samples preparation

DNA extraction and purification method - CTAB method 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 Na. Cl 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 washed in 70% ethanol, vacuum dried and resuspended in 100 μl MQ water. Quantification of the extracted DNA by spectrophotometric method Thermo Scientific Nano. Drop 8000

PCR analyses Amplification was performed in a Corbett RESEARCH Thermal Cycler, folowing the indications from literature. PCR reagents: Go Taq Green Master Mix PCR kit from Promega 2 X. 20 pmol of each primer. different concentrations of DNA template. in a final volume – 25 µl. Amplicons were analyzed by electrophoresis on 2% agarose gel (Promega, USA) and visualized in Ethidium Bromide (0. 4 ng/ml) presence.