Gotaq g2 hotstart enzyme

According to BgTEP2 cDNA sequence, a couple of primer (F1: 5′-ACTACGGAGGCAGTGATGC-3′; R1: 5′-GATAGTATCTGGTACTGTTGC-3′) designed in non-variable parts of the sequence and framing the highly variable region was used to detect all the different alternatively spliced isoforms of BgTEP2 from snail tissues prepared as described above. Then, 1 µL of a 1:30 dilution of cDNA was used as template amplification with primers F1 and R1 using the GoTaq G2 HotStart enzyme (Promega, Madison, Wisconsin, USA) in a 50 µL final reaction, according to the manufacturer’s instructions. PCR reaction was performed in a Biometra TOne thermocycler (Analitik Jena AG, Jena, Thuringia, Germany), using the following cycling program: Initial denaturation 3 min at 95 °C, 45 cycles of amplification (denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 20 s), final elongation of 2 min at 72 °C. Then, 20 µL of the resulting PCR were analyzed by electrophoresis on a 2% agarose gel. Another couple of primer targeting the cDNA coding for the ribosomal protein S19 was used as a positive control of PCR amplification (S19F: 5′-TTCTGTTGCTCGCCAC-3′; S19R: 5′-CCTGTATTTGCATCCTGTT-3′).

In order to characterize coding sequences, total RNA from a pool of 10 snails was extracted with TRIzol reagent (ThermoFisher Scientific, Paris, France) according to the manufacturer’s instructions and subsequently reverse transcribed to first strand cDNA using Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (ThermoFisher Scientific, Paris, France).
Four Glabralysin gene fragments (Gla1Aa1, Gla1Aa2, Gla1Ba1, Gla1Ca1) were obtained by running polymerase chain reaction (PCR), using GoTaq G2 Hot Start enzyme (Promega, Lyon, France). Briefly, 2.4 µL MgCl₂ (1.5 mM), 8 µL Gotaq Buffer 5×, 0.8 µL dNTPs (10 mM), 0.2 µL Gotaq enzyme (1 unit), and 0.5 µL of primers (100 µM), for a total reaction volume of 40 µL. Primer sequences are shown in Table S1. The amplification cycling conditions were as follows: initial denaturation step at 95 °C for 6 min, followed by 40 amplification cycles composed of 30 s denaturation at 95 °C, 30 s primer annealing step at 51 °C and 1 min elongation at 72 °C. PCR reaction was ended by a final elongation step of 5 min at 72 °C. Amplicons were sequenced by Sanger method. Glabralysin gene structure (exon/intron) was determined by comparing cDNA sequences obtained from sequencing to the B. glabrata BglaB1.6 genome assembly using VectorBase website [49 ].