Plasmid extraction kit

Lyophilized mycelium was ground into a fine powder and dispersed (in the case of DNA for use in PCR) in extraction buffer as described by Cenis [81 (link)]. DNA for Southern hybridization experiments was prepared following the protocol of Doyle and Doyle [82 ]. Plasmid DNA was extracted using the Genomed plasmid extraction kit (Genomed, Löhne, Germany). Total F. fujikuroi RNA was isolated using the RNAgents total RNA isolation kit (Promega, Mannheim, Germany). For Southern analysis, genomic DNA was digested to completion with appropriate restriction enzymes, fractionated in 1.0% (w/v) agarose gels, and transferred to nylon membranes (N+, Amersham). DNA probes were randomly labelled and hybridizations were carried out overnight at 65°C.

Lyophilized mycelium was ground into a fine powder and dispersed (in the case of DNA for use in PCR) in extraction buffer as described by Cenis (1992) (link). Plasmid DNA was extracted using the Genomed plasmid extraction kit (Genomed, Löhne, Germany). Total F. fujikuroi RNA was isolated using the RNAgents total RNA isolation kit (Promega, Mannheim, Germany). For Northern blot analysis, samples of 20 μg of total RNA were transferred to Hybond-N+ membranes after electrophoresis on a 1% (w/v) agarose gel containing 1% (v/v) formaldehyde. DNA probes were labeled with ³²P isotopes using the random oligomer-primer method (Sambrook et al., 1989 ). Hybridizations were carried out overnight at 65°C (Church and Gilbert, 1984 (link)). The following probes were used and amplified with the indicated primer combinations (see Supplementary Table S1 for primer sequences): BIK2 (bik2-F/bik2-R), CPS/KS (cps/ks-RT-for/cps/ks-RT-rev), NIAD (NIAD-WT-F/NIAD-WT-R), NIIA (NIIA-WT-F/NIIA-WT-R), NIRA (NIRA-WT-F/NIRA-WT-R), NRTA (NRTA-WT-F/NRTA-WT-R).