Chloroform isoamylalcohol

The total community DNA from 0.5g of each wood sample was isolated using a modified CTAB-protocol [36 ] as described in Hoppe et al. [37 (link)]. Briefly, 900 μl of CTAB was added to the sample and nucleic acids were separated from proteins and cell debris by adding 500μl of 24:1 chloroform: isoamyl alcohol (Carl Roth, Karlsruhe, Germany) followed by another chloroform step (Carl Roth). DNA was precipitated and washed twice with 99% ethanol (Merck, Darmstadt, Germany). Dried DNA pellets were dissolved in 100 μl molecular grade water (AppliChem, Darmstadt, Germany).

RNA isolation was performed by the phenol as follows extraction based on a previously described protocol^{56 (link)}: overnight 10 mL cultures were centrifuged at 4 °C and 4816×g for 15 min and immediately used for RNA isolation. After removal of the medium, cells were suspended in 0.5 mL of ice-cold TE buffer (pH 8.0) and kept on ice. All samples were divided into two 2 mL screw-capped tubes containing 0.5 g of zirconium beads, 30 μL of 10% SDS, 30 μL of 3 M sodium acetate (pH 5.2), and 500 μL of Roti-Phenol (pH 4.5−5.0, Carl Roth GmbH). Cells were disrupted using a FastPrep-24 apparatus (MP Biomedicals) at 5500 r.p.m. for 45 s and centrifuged at 4 °C and 9400×g for 5 min. 400 μL of the water phase from each tube was transferred to a new tube, to which 400 μL of chloroform−isoamyl alcohol (Carl Roth GmbH) was added, after which samples were centrifuged at 4 °C and 18,400×g for 3 min. 300 μL of the aqueous phase was transferred to a new tube and mixed with 300 μL of the lysis buffer from the high pure RNA isolation kit (Roche). Subsequently, the rest of the procedure from this kit was performed according to the manufacturer’s protocol, except for the DNase incubation step, which was performed for 45 min. The concentration and integrity of cDNA was determined using Nanodrop-1000 Integrity and concentration of the isolated RNA was checked on a NanoDrop 1000.

Plasmid DNA was isolated according to the protocol of Anderson and McKay [34 (link)] with the following modifications: after the addition of lysozyme (10 mg/mL, SERVA Electrophoresis GmbH, Heidelberg, Germany), 20 µL RNase A (100 mg/mL, Qiagen GmbH, Hilden, Germany) was added to the reaction mixes, which were incubated at 37 °C for 60 min. Phenol, which was saturated with TE-buffer and chloroform-isoamyl alcohol (both Carl Roth GmbH & Co. KG, Karlsruhe, Germany), was used for the protocol. Purified plasmid DNA was dissolved in 30 µL of TE-buffer and analyzed by electrophoresis on 0.8% (w/v) agarose gels.