Cdp star

BAC filters were hybridized with probes labeled and detected with CDP-Star (GE Healthcare) or DIG-labeling kit (Roche). Positive signals were confirmed by direct PCR with probe-specific primers on the individual clones. BACs of true positives were column-purified from 5 ml cultures, and end-sequenced with M13 Forward and Reverse primers. Each end sequence is designed for a pair of specific primers, which was used to determine the relative locations of overlapping BACs. Primer pairs from both distal ends were selected to label probes for the next round of chromosomal walking. Inverse PCR (Sambrook and Russell 2001 ) and TAIL-PCR (Liu and Whittier 1995 (link)) were performed to identify flanking sequences when no additional BAC clones could be identified. GpMTD1 probes were the same as those used for the GpMTD1 DNA gel-blot analysis (Hamaji et al. 2009 (link)). The PCR product of GPLEUFATG and GPLEURTAA was labeled to screen for LEU1S alleles (Supplemental Material, Table S1). Two gametologs (WDR57 and DRG1) and their flanking regions, which were not obtained in the MT+ BAC assembly, were obtained by genomic PCR using specific primers based on the MT– alleles. The linkage of MT+ DRG1 was determined by recombination scores as described below. Abundant repetitive regions flanking the MT assembly prevented further chromosomal walking to connect directly to autosomal sequences.

Orientation of the region between IR₁ and IR₂ [A(α) or I(a)-type] were determined by PCR as previously described with the primers listed in Supplementary Table S2^{8 (link)}. For Southern blot analysis, O. polymorpha genomic DNA was prepared using a standard protocol. Briefly, DNA was digested with EcoRI restriction enzyme before electrophoresis. A standard protocol was used for blotting and hybridization. DNA probes were prepared, and detection was performed using the AlkPhos direct labeling and detection system with CDP-Star (GE Healthcare, Pittsburgh, PA, USA). Signal intensities were quantified with ImageJ version 1.47 (National Institute of Health, Bethesda, MD, USA) and Photoshop (Adobe Systems, San Jose, CA, USA) was used to mount the images.

Following 2D electrophoresis, gels were washed sequentially in depurination buffer (0.125 M HCl), denaturation buffer (0.5 M NaOH, 1.5 M NaCl) and neutralisation buffer (0.5 M Tris–HCl, 1.5 M NaCl pH 7.5) with washes in ddH₂O in-between each buffer. DNA was transferred onto Hybond-N+ membrane (GE Healthcare) by capillary action in 20× SCC (3 M NaCl, 350 mM NaOC trisodium citrate pH 7.0). Membranes were cross-linked using a UV Stratalinker 1800 (Stratagene) at 1200 J/m and subsequently blocked in hybridisation buffer (5× SSC, 5% Dextran sulphate (Sigma-Aldrich, D8906) 0.2% Tropix I-Block (Applied Biosystems, T2015), 0.1% SDS) for at least 1 h at 60°C.
Catenated pRS316 plasmids or replication intermediates from pRS426-RFB were probed with DNA amplified from pRS316 (probing specifically for the URA3 gene). Labelling and detection used random prime labelling incorporating fluorescein tagged dUTP (Roche). Following probing, hybridized fluorescein tagged dUTP was detected with alkaline phosphatase tagged anti fluorescein Fab fragments (Roche), revealed with CDP-Star (GE Healthcare) and non-saturating exposures acquired on an ImageQuant LAS4000 system (GE Healthcare). Densitometry analysis was carried out using ImageQuant TL software.

Genomic DNA (30 µg), extracted by the cetyl trimethylammonium bromide (CTAB) method²⁶ , was digested with HindIII, resolved by agarose gel electrophoresis, and blotted onto a Hybond-N⁺ membrane (GE Healthcare, UK). A GFP gene-specific probe was directly labelled with alkaline phosphatase using the AlkPhos Direct labelling kit (GE Healthcare, UK). Hybridisation, washing and chemiluminescent detection with CDP Star were performed as recommended by the supplier (GE Healthcare).

DNA gel blot analysis9 (link) was performed as below: ∼3 μg of genomic DNA was digested with appropriate restriction enzyme(s) (see Supplementary Figs 2, 4 and 7), run on 0.7% (w/v) SeaKemGTG agarose (BME, Rockland, ME, USA), and transferred to a Hybond N⁺ nylon membrane (GE Healthcare, Chicago, IL, USA). Probe labelling, hybridization and detection were performed using the AlkPhos direct labelling and detection system with CDP-Star (GE Healthcare) according to the supplier's instructions. Primers used for probe amplification are provided in Supplementary Table 1.

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).