Dig high prime labeling and detection starter kit 2

Genomic DNA was extracted using the gDNA Midi Kit (Zymo Corp, Irvine, CA). 7–15 µg genomic DNA from each iPSC line were digested overnight with Eco32I or EcoO109I (Fermentas) and resolved by electrophoresis on 0.8–1.0% agarose gels at 16–22 V for 6–10 h. DNA was transferred using the Whatman kit (Schleicher and Schuell, Keene, NH) and nitrocellulose membrane (Hybond-N; GE Healthcare, Piscataway, NJ). The Turboblotter transfer apparatus (Schleicher and Schuell) was used following the manufacturer's instructions. A DIG-labeled GFP probe generated by the DIG High Prime Labeling and Detection Starter Kit II (Roche, Indianapolis, IN) was then hybridized to the membrane following the manufacturer's instructions.

RNA was isolated from cells grown under the specified conditions, using either a modified hot-phenol extraction method [5] (link) or the RNeasy mini kit (QIAGEN). RNA concentration and quality were assessed by determining (in 0.1 N NaOH) the optical density at 260 nm (OD₂₆₀) and the 260 nm/280 nm ratio, respectively. Electrophoresis was carried out essentially as previously described [5] (link) loading 5 µg of total RNA per sample. To detect yhiM-specific mRNA, northern blots were hybridized with a 1050-bp yhiM probe obtained by PCR amplification using plasmid pCRIIyhiM (see below) as template and the oligonucleotide pair yhiM_for and yhiM (Bgl)_rev (Table 2). The yhiM-specific probe was labelled and hybridized with the DIG High Prime Labeling and Detection Starter Kit II (Roche), according to manufacturer instructions.
Primer extension analysis was performed with the oligonucleotide yhiM_revRT (Table 2), which anneals 286 nt downstream the putative GTG start codon, and using total RNA (7.0 µg) extracted from either E. coli MC4100 or E. coli MG1655 grown to the stationary phase in LB-MES, pH 5.5. The entire procedure was essentially as previously described [5] (link).