Whatman turboblotter transfer system

A published protocol was used (He and Green, 2013 (link)). In brief, 15 microG of total RNA was mixed with 2x RNA-loading dye (New England Biolab), incubated at 65℃ for 20 min, and run on a denaturing RNA gel (1.2% agarose, 1x MOPS buffer) at 90 V for 5 h, along with the ssRNA ladder (New England Biolabs). The RNA was transferred onto a nylon membrane using Whatman TurboBlotter transfer system (GE Healthcare) according to the manufacturer’s instruction. The membrane was then UV crosslinked. DNA probes were prepared using the PCR DIG Probe system (Millipore Sigma). cDNA from a human cell line was amplified by PCR using the primers for ONECUT2 (5′ GAG​TCT​GCC​CAA​CTA​CGG​TC-3′ and 5′- GCG​TTT​GCA​CGC​TGC​C-3′) and GAPDH (5′-CAG​CCT​CAA​GAT​CAT​CAG​CAA​TG-3′ and 5′-AAA​TGA​GCT​TGA​CAA​AGT​GGT​CG-3′).

Southern blotting was carried out on a single mtpA transformant for confirmation of mtpA gene disruption. As shown in Fig. 2a, the hybridization probe was prepared by amplifying a part of the hph marker gene with primers 11–12 (Table 3) and labeled using a North2South chemiluminescent detection kit (Thermo Fisher Scientific). Genomic DNA of the transformant was digested by EcoRI and HindIII and hybridized with the probe. Southern blotting was then performed using Whatman Turboblotter transfer system (GE healthcare life sciences) and detected by using a Pierce Chemiluminescent Nucleic acid detection module Kit (Thermo scientific). The blot was imaged in Thermo ECL imager.

The hybridization probe for the southern blot analysis of the TDR transformants was designed to consist of a single fragment containing FADO, TprC terminator, tef1 promoter and FAR sequence, originating from the previously constructed expression vector (Fig. 3a). The DNA probe was biotin labeled by using Pierce North2South Biotin Random Prime Kit (Thermo Fisher Scientific, Rockford, IL, USA) according the manufacturer’s protocol. Genomic DNA of the transformants was extracted as described above and digested with restriction enzymes BamHI, PstI, and NdeI (all purchased from Thermo Scientific, Rockford, IL, USA). Southern blotting was carried out using Whatman Turboblotter transfer system (GE Healthcare Life Sciences, Pittsburg, PA, USA) and the nucleic acid detection was carried out by using Pierce Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Rockford, IL, USA), both according to the manufacturer’s protocol.