Mce 202 multina

Small pieces of tail fin were cut and added to 20 µL lysis buffer (0.1 M Tris-HCl, 0.2 M NaCl, 0.1% SDS, 5 mM EDTA). After 5 min incubation at 95°C, 80 µL H₂O were added. After centrifugation, 1-µL aliquots of supernatant were used for genomic PCR with KOD FX Neo (TOYOBO) and appropriate primers (Table 4). Candidate G0 fish were crossed with wild-type counterparts, and fertilized eggs were analyzed for fluorescence of RCC1-mCh or RCC1-mACF, 1 or 2 days after fertilization. Genomic DNA was extracted from some fluorescence-positive F1 embryos, and genomic PCR was performed. Proper knock-in of the construct was confirmed by direct sequencing of PCR products. Other positive embryos from the same G0 fish were reared, and F1 male and female fish were mated to obtain a homozygous F2 generation. PCR products were analyzed by normal gel electrophoresis or using an automatic microchip electrophoresis system (MCE-202 MultiNA: Shimazu, Kyoto, Japan).
To select the EGFP-α-tubulin knock-in strain (Supplementary Fig. 1b), embryos with EGFP fluorescence in their eyes and throughout their bodies were raised as G0 founders and were crossed with wild-type medaka to obtain a stable F1 transgenic line. Insertion of the KI vector at the target site was examined by PCR using specific primers for the KI vector and the promoter of α-tubulin (upstream of gRNA target site).

A 215 bp fragment containing the target sites of CjChlI was amplified using primers p#468_f and p#469_r (Supplementary Table S1). PCR amplification was performed under the following conditions: 94 °C for 2 min; 40 cycles at 98 °C for 10 s, 65 °C for 30 s, and 68 °C for 20 s; and final extension at 68 °C for 7 min. To ensure full heteroduplex formation, a denaturation and re-annealing procedure was performed on the PCR products as follows: 95 °C for 5 min; 95–85 °C, ramping at − 2 °C/s; and 85–25 °C at − 0.1 °C. Reannealed products were analyzed using a microchip electrophoresis system (MCE-202 MultiNA, Shimazu, Kyoto, Japan) with the DNA-500 reagent kit (Shimazu).

A 285-bp fragment including the genomic target sequence of the sgRNA was amplified using the primers DJ1-FW2 and DJ1-RV1 (supplementary material Table S1). The reaction mixture contained 2 µL of genomic DNA template, 1× reaction buffer, 0.8 mM of each dNTP, 1.5 mM of MgCl₂, 0.2 mM of each primer, and 0.5 unit of HybriPol DNA Polymerase (Bioline, London) in a total volume of 20 µL. The cycling conditions were as follows: one cycle at 95°C for 2 min, followed by 30 cycles of 95°C for 20 sec, 58°C for 30 sec, and 72°C for 30 sec. The resulting product was precipitated with ethanol for buffer exchange and was digested at 37°C for overnight in 10 µL of the solution containing 1× L buffer and 2 units of the AluI restriction enzyme. After inactivation at 80°C for 10 min, the digested fragments were analyzed using a microchip electrophoresis system (MCE-202 MultiNA; Shimazu) with the DNA-500 reagent kit. The molar concentrations of both digested and undigested fragments were quantified using the MultiNA Viewer software. The mutation rate was calculated as the ratio of the undigested fragment to the sum of the undigested fragment and the larger digested fragment as described previously (Ansai et al., 2013 (link)).

Genomic DNA of LAB and yeasts was extracted as described above. Biotyping of LAB was carried out through Random Amplification of Polymorphic DNA (RAPD)-PCR using primers P4, P7 (De Angelis et al., 2001 (link)), and M13 (Siragusa et al., 2009 (link)). Yeasts were biotyped through RAPD-PCR using primers M13m and RP11 (Del Bove et al., 2009 (link)). RAPD-PCR profiles were acquired by the microchip electrophoresis system MCE-202 MultiNA (Shimadzu Italia s.r.l., Milano, Italy), as previously described (Minervini et al., 2016 (link)). The similarity of the electrophoretic profiles was evaluated by the Pearson product moment correlation coefficient (r) and using the Unweighted Paired Group Mathematic Average (UPGMA) algorithm. Identification of bacterial and yeast strains was performed as described above.

The genotypes were confirmed by PCR using genomic DNA (gDNA). gDNA was extracted, as described previously [31 (link)]. The DNA fragments containing each target site were amplified by PCR using KOD-FX and specific primers (Abca1a: 5’-TACTAGATAACATTGAGGCAG-3’ and 5’-GCTGCGGAGTATAAGTCGCAC-3’; Abca1b: 5’-TGTGCATTGAGGAGGAACCCG-3’ and 5’-GTCCTTCCCGAGGATGTAAGC-3’; and Abca1c: 5’-GTTTGTGTGGAGGAGGAGCCTG-3’ and 5’-TCCCAAGATGTAGGCGGTGCCAG-3’) or BIOTAQ and specific primers (Dmy: 5’-CCGGGTGCCCAAGTGCTCCCGCTG-3’ and 5’-GATCGTCCCTCCACAGAGAAGAGA-3’). The resulting amplicons were analyzed using a microchip electrophoresis system (Shimadzu, model: MCE-202 MultiNA) with a DNA-500 reagent kit for Abca1a, Abca1b, and Abca1c or 1% agarose electrophoresis for Dmy.