8. Nucleic acid manipulation
Fungal biomass from liquid cultures, wheat roots colonized byTgam and fungal mycelium from the Tgam - root interaction control plates were ground in liquid N2 using pre-chilled mortar and pestle. Samples containing 100 mg of powder were used for total RNA extraction using the RNeasy® Plant Mini Kit (Qiagen), according to the manufacturer’s instructions. Wheat spikes colonized by the fungus were ground in liquid N2 using pre-chilled mortar and pestle. Samples containing 300 mg of powder were used for total RNA extraction according to the method described by Logemann, Schell and Willmitzer (1987). RNA samples were treated with DNase I (DNase I Amplification Grade, AMPD1 Sigma-Aldrich) for gDNA removal, according to the manufacturer’s instructions. A total of 400 ng of RNA were used for cDNA synthesis using Maxima First Strand cDNA synthesis kit (K1642 Applied biosystems) according to the manufacturer’s instructions.