Speedcycler2

To analyse transcription of genes from the polycistronic messenger RNA of the ATNT16 P2A_P2A strain RNA was isolated using the MasterPure-Yeast RNA Purification Kit (Epicentre) from mycelium cultivated for 24 h in the absence or presence of 15 µg/ml doxycycline or pre-grown for 18 h without doxycycline and further cultivated for 6 h after addition of doxycycline. After a DNase treatment (TURBO DNase; ThermoFisher) RNA was transcribed into cDNA as previously described [19 (link)]. For normalisation of cDNA levels in the respective samples, serial dilutions were used for amplification of the A. niger actin gene using oligonucleotides 25 and 26. These primers span an intron region, which allows visualisation of a band shift from cDNA compared to genomic DNA (gDNA) and confirms the absence of contaminating gDNA in cDNA samples. For amplification of the melA gene oligonucleotides 15 and 16, for tyrP oligonucleotides 27 and 28 and for the tdTomato gene oligonucleotides 20 and 29 were used. PCRs of 30 cycles were performed in a SpeedCycler² (Analytik Jena) using Phire Hot Start II polymerase (Thermo Scientific).

Genomic DNA was extracted using the DNeasy Plant Minikit (Qiagen, Germany). The quantity and quality were determined by spectrophotometry (Nanophotometer, IMPLEN, USA). Genomic DNA extracted from leaf samples were considered yielding DNA of good quality with A₂₆₀/A₂₈₀ ratio between 1.700 and 1.900 (Sambrook & Russel 2001 ). PCR amplification of the trnL-trnF intergenic spacer region was carried-out using primer E: 5′-GGT TCA AGT CCC TCT ATC CC-3′, and primer F: 5′-ATT TGA ACT GGT GAC ACG AG-3′ (Taberlet et al. 1991 (link)), while the nuclear ribosomal ITS region was amplified using primer ITS-p5: 5′-CCT TAT CAY TTA GAG GAA GGA G3′, and ITS-u4: 5′-RGT TTC TTT TCC TCC GCT TA-3′ (Cheng et al. 2015 (link)). PCR reaction was prepared in a 25 μl volume containing 12.5 μl of PCRBioTaq Mix Red (PCR Biosystems, UK), 0.4 μM of each primer, 5 – 25 ng of genomic DNA. PCR amplification was carried-out in a SpeedCycler² (Analytik Jena, Germany) as follows: denaturation at 95°C for 1 min, 40 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 15 s, and a final extension of 72°C for 3 min. PCR products were sent for direct sequencing at First BASE Laboratories Sdn Bhd, Selangor, Malaysia, on an ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems, USA).