Bluejuice gel loading buffer

Paraformaldehyde (PFA) and chloroform were obtained from Merck Milipore, USA. NaHPO₄, NaH₂PO₄, and isopropyl alcohol were obtained from Sigma-Aldrich, Germany. TRIzol reagent (Invitrogen), SuperScript™ IV First-Strand Synthesis System, SuperScript™ IV Supermix, 1 kB Plus DNA Ladder, 10X Blue Juice™ Gel Loading Buffer, Agarose powder, and 10X TBE Buffer were purchased from Thermo Fisher Scientific, USA. DORMINAL 20% (200 mg Pentobarbital Sodium) (Alfasan, Holland), Ethanol denatured (HmbG, Germany), NMR buffer (KH₂PO₄ and sodium azide), and Trimethylsilylpropanoic acid (TSP) were used as a reference signal for NMR spectra. All chemicals and reagents used were of analytical grade.

Various amounts of HK peptides were mixed with 1 μg of mRNA and incubated for 30 minutes at room temperature. Specifically, the following HK/mRNA ratios (w/w) were prepared in water: 1/2; 1/1; 2/1, 4/1 and 8/1. After 30 minutes, the HK polyplex was loaded onto the gel (20 μl; 1% agarose, Sigma‐Aldrich; 10X BlueJuice Gel loading buffer, Thermo Fisher Scientific), and electrophoresis was then carried out at a constant voltage of 75 V for 30 min in Tris‐acetate‐ethylenediaminetetraacetic acid (TAE) buffer (Quality Biologicals, Gaithersburg, MD, USA). The mRNA was stained with Sybr Gold Nucleic Acid dye (SG, 1X) (Thermo Fisher Scientific) for 30 minutes before exposure to the ultraviolet imager (ChemiDoc Touch; Bio‐Rad, Hercules, CA, USA).

Genetic diversity of the C. truncatum collection was examined via RAPD. Firstly, a group of 12 primers (Operon Technologies, Alameda, CA, USA) which produced clear, consistent, polymorphic bands were selected from a larger group of 64 primers, using a six-isolate subset of the collection, in duplicate. Subsequently, PCR reactions of all 54 isolates were individually performed with the 12 primers with the most stable polymorphic patterns (Table 3). Reactions were performed on a final volume of 13 µL consisting of 3.42 µL milli-Q water, 1.3 µL PCR buffer 10×, 1.04 µL de Bovine Serum Albumin (BSA), 1.04 µL dNTPs, 0.20 µL Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 3 µL primer (10 ng/µl), and 3 µL of DNA (3 ng/ µL). PCR was conducted on a GeneAmp^® System 9700 thermocycler with reaction conditions consisting of an initial 5 min denaturation at 92 °C, followed by 40 cycles (1 min denaturation at 92 °C, 1 min annealing at 35 °C, 2.5 min extension at 72 °C), and a 5 min final extension at 72 °C. Blue Juice^® gel loading buffer (Invitrogen, Carlsbad, CA, USA) was added to the PCR products (2.0 µL per sample) and samples electrophoresed in 1.5% agarose gels, in 1× TBE buffer, at 160 V for 90 min. Gels were stained with 1.5% ethidium bromide and photographed under UV light. Amplicon sizes were estimated using High DNA Mass Ladder 1KB (Gibco BRL, Gaithersburg, MD, USA).