FastDigest Eco32I

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	After amplification, the ETAS-PCR/Vgsc-1014 multiplex products were digested with FastDigest Eco32I restriction enzyme (Thermo Scientific, United Kingdom) in a final reaction volume of 30 µl including 10 µl of the PCR product, 2 µl of 10× FastDigest Green buffer, 1.5 µl of FastDigest enzyme and 17 µl distilled water. The reaction was incubated at 37 °C for 10 min. Finally, 10 µl of digestion reaction was loaded on a 3% agarose gel.

Protocol tips

After amplification, the ETAS-PCR/Vgsc-1014 multiplex products were digested with FastDigest Eco32I restriction enzyme (Thermo Scientific, United Kingdom) in a final reaction volume of 30 µl including 10 µl of the PCR product, 2 µl of 10× FastDigest Green buffer, 1.5 µl of FastDigest enzyme and 17 µl distilled water. The reaction was incubated at 37 °C for 10 min. Finally, 10 µl of digestion reaction was loaded on a 3% agarose gel.

Publication protocol

The ETAS-PCR/Vgsc-1014 multiplex reaction was performed in a total volume of 25 µl containing 10 ng of gDNA, 200 µM each dNTP, 1× PCR buffer, 0.2 units HotStartTaq DNA polymerase (Qiagen), 0.6 µM universal reverse primer Vgsc-1014/R, 0.35 µM of the specific forward primers (Vgsc-1014/F-T and Vgsc-1014/F-C) and 0.3 µM of the Vgsc-1014/F-A primer (Table 1). After initial denaturation at 95 °C for 15 min, amplification was performed for 40 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s, followed by a final extension step of 72 °C for 10 min.After amplification, the ETAS-PCR/Vgsc-1014 multiplex products were digested with FastDigest Eco32I restriction enzyme (Thermo Scientific, United Kingdom) in a final reaction volume of 30 µl including 10 µl of the PCR product, 2 µl of 10× FastDigest Green buffer, 1.5 µl of FastDigest enzyme and 17 µl distilled water. The reaction was incubated at 37 °C for 10 min. Finally, 10 µl of digestion reaction was loaded on a 3% agarose gel.

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