Model 785 vacuum blotter

The total RNA preparation (500 ng/μL) was denatured at 68 °C for 15 min and fractionated by electrophoresis at 100 V for 10 min on a 1.2% (w/v) agarose gel in 1× 3-(N-morpholino) propane sulfonic acid (MOPS) buffer. RNA was then transferred to a nylon membrane (Biodyne; Pall Corp., Port Washington, NY, USA) using a vacuum blotting apparatus (Vacuum Blotter Model 785; Bio-Rad Laboratories, Hercules, CA, USA) for 30–90 min at 5 inches mercury (Hg), UV-crosslinked at 120,000 µJ/cm² in a UV crosslinker (UVP CX-2000; BM Equipment Co., Ltd., Tokyo, Japan), and hybridized with digoxigenin (DIG)-labeled cRNA probes as described previously [61 (link)]. DIG-labeled cRNA probes for tomato genes EXPASIN 2 (slEXPA2; DDBJ accession no., AF096776), and chalcone synthase (TCHS2; DDBJ, accession no., X55195) were prepared from recombinant plasmid DNAs containing a partial sequence of the gene in pBluescript II SK(–) under the control of either T7 or T3 RNA polymerase promoter constructed in this study. Hybridized signals were visualized using the ChemiDoc-XRS (Bio-Rad Laboratories, Hercules, CA, USA) imaging system and quantified using the Quantity One (version 4.6.2) software package.

RNA samples were boiled at 95 °C for 8 min, then cooled to room temp, spun briefly and loaded onto the gel. Gel was run at 100 V for ~2.5 hr. Gels were imaged on a Typhoon imager to assess RNA quality, then transferred to a Amersham Hybond N+charged nitrocellulose membrane by a BioRad Model 785 Vacuum Blotter following the manufacturer’s instructions for transferring RNA, with the alterations of prewetting the membrane with 10 x SSC only and maintaining vacuum between 10–15 inHg. Transfer proceeded for 2 hr. Following transfer, the membrane was carefully removed from the vacuum blotter and placed face up on paper towel for UV crosslinking in a Stratagene UV Stratalinker 2400 on the automatic setting (120 mJ) three times.

Approximately 10 μg of genomic DNA per lane was digested with the restriction enzymes Eco RI, Pst I, or Sac I (TaKaRa) and electrophoresed on a 0.7% agarose gel. DNA samples were then transferred to a Hybond‐N+ nylon membrane (GE Healthcare, Buckinghamshire, UK) using a Model 785 vacuum blotter (Bio‐Rad, Hercules, CA, USA). DNA was fixed to the membrane using an XL‐1500 UV cross‐linker (Spectroline, NY, USA). As a hybridization probe, exon 1 of the barfin flounder rh2‐b gene was used (previously described in Kasagi et al., 2015). Amplified cDNA was labeled with an Alkphos‐Direct labeling kit (GE Healthcare), and hybridization was performed according to the manufacturer's instructions. Stringent washing was conducted at 60°C in primary buffer wash (per the manufacturer's instructions), which contained 0.2 M of NaCl and 0.1% of SDS, which allowed approximately 20% mismatch (Sambrook & Russell, 2001). Signals were detected using CDP‐Star (GE Healthcare) and Hyperfilm ECL (GE Healthcare).

Genomic DNA (10 ug) cleaved with SacI was transferred to Amersham Hybond-N⁺ membrane (GE Healthcare, Chicago, IL, USA) by 10 × SSC using Model 785 Vacuum Blotter (Bio-Rad, Hercules, CA, USA). [α-³²P] dCTP-labeled hpt, gus, gfp, and bar fragments with Amersham Rediprime II Random Prime Labelling System (GE Healthcare, Buckinghamshire, UK) were used as hybridization probes. Hybridization and washing methods were according to Sambrook et al. [38 ]. After washing, the membranes were exposed to a phosphor screen for 5–12 h and scanned on Typhoon FLA 9500 (IP: 635 nm, PMT: 500 V, Pixel size 200 μm). Probe primer sequences are presented in Table S2.