Amersham alkphos direct labeling and detection systems

Cells were grown in YPD medium to mid-exponential phase (OD₆₀₀ = 0.4 to 0.7), and collected by filtration. RNA was prepared by a hot phenol method (Collart and Oliviero, 2001 (link)). Northern blot analysis was performed as described previously (Morohashi et al., 2006 (link)) with following modifications: STE11 and FLO8 PCR fragments (Pattenden et al., 2010 (link)) were used as probes that were labeled with alkaline phosphatase using Amersham AlkPhos Direct Labeling and Detection Systems (GE healthcare). Blots were exposed to HyBlot CL Autoradiography Film (Denville Scientific). Signal intensity was quantified by using ImageJ.
Primers for Northern probe (Pattenden et al., 2010 (link)):
o-STE11-11: GAA GGA GTT ACA TCA TGA GAA CAT TGT TAC
o-STE11-12: GTG TGC ATC CAG CCA TGG ATG CTG CAG CAA
o-FLO8-13: GAC GCT CAG AAG CAA AGA AGT TCT AAG GTA
o-FLO8-14: CTC AAC ACG TGA CTT CAG CCT TCC CAA TTA

Verticillium dahliae was grown in CDB liquid media for 10 days, then mycelia were collected and ground in liquid nitrogen. The DNA extraction method followed the protocol of Al-Samarrai and Schmid (2000) (link). Genomic DNA of V. dahliae (∼20 μg) was digested by restriction enzyme EcoRV overnight at 37°C, and the digested DNA was separated in a 0.8% agarose gel for 6 h at 70 V. The DNA was transferred to a nylon membrane following the protocol of Maruthachalam et al. (2011) (link). Hybridization and detection were conducted with Amersham AlkPhos Direct Labeling and Detection Systems (GE Healthcare Life Sciences, Mississauga, ON, Canada). The signal was visualized on X-ray film (Kodak, Rochester, NY, USA).