**2.5 The extraction of gDNA from maize tissues and** *Aspergillus flavus*

A 100 mg of each of the following samples: control healthy maize tissues, infected and *A. flavus* KSM014 mycelia was used to extract DNA following the method of Möller et al. [16] with some modification. Briefly, 100 mM Tris pH 8.0, 2% SDS, modified TES buffer, 2% (w/v) polyvinylpyrrolidone (PVP) and 10 mM EDTA was prepared. 5 μl RNase (10 mg/ml) and 450 μl of TES buffer was added to microtube (2 ml) containing the tissues and thereafter, homogenized by vortexing for 15 min or with a microtube pestle. Twenty microlitres of Proteinase K (1 μg/μl) was added and vortexed for 1 min, thereafter, incubated for 1 h at 60°C. Seventy microliters of 10% CTAB (0.1 vol.) and 160 μl of 5 M NaCl (0.3 vol. was added and incubated at 65°C for 10 min). Seven hundred and fifty microliters of chloroform/ isoamyl-alcohol (24:1) was added, vortexed for 5 min, incubated on ice for 30 min and centrifuged at 14,000 rpm for 10 min. The aqueous phase was transferred cautiously onto a new microtube (2 ml) and 300–350 μl isopropanol (0.55 vol.) added and left to stand for 30 min at RT after mixing gently for 30 s. The mixture was centrifuged for 10 min at 14,000 rpm. Supernatant was cast-off, the pellets rinsed with chilled 700 μl of 70% ethanol twice and centrifuged again at 14,000 rpm for 2 min after mixing gently. The pellets were air dried and dissolved in 40 μl TE buffer (10 mM Tris-Cl pH 8, 1 mM EDTA pH 8) or nuclease free water after discarding the ethanol. The integrity of DNA was assessed on a 1% agarose/EtBr gel and the concentration quantified on a Nano-DropTM 1000 spectrophotometer (Nano Drop Technologies, USA). DNA was diluted to 10 ng/μl for further analysis.
