Zeta probe gt

Exponentially growing yeast cells were shifted from galactose to glucose containing medium and grown for 8 hours. RNA was isolated from 25 OD_{600 nm} units of exponentially growing yeast cells using the hot phenol method described previously [23 (link)]. Roughly 2.5 μg of total RNA from each strain was separated on a 1,5% agarose-formaldehyde gel. Before loading, ethidium bromide was added to each RNA sample at a final concentration of 10 μg/ml. After electrophoretic separation, the RNA was detected using a Gel Doc^™ Ez Image System (BioRad) and quantified using the Image Gauge V3.3 software. The RNA was subsequently blotted to a nylon membrane (Zeta-Probe GT, Bio-Rad) by capillary transfer and UV cross-linked in a Stratalinker (Stratagene). The oligonucleotide probe (5′ GCTCTCATGCTCTTGCC 3′), specific for the S. cerevisiae ITS1 between cleavage sites D and A₂, was end-labeled using γ-³²P ATP (Perkin Elmer) and T4 Polynucleotide Kinase (Thermo Scientific) and purified using the QIAquick Nucleotide Removal kit (Qiagen). Membranes were hybridized in 0.5M sodium phosphate, pH 7.2, 7% SDS at Tm—9°C, washed and analysed with a Fuji Bio-Imaging analyzer FLA3000 using the Image Gauge V3.3 software.