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.