Eco47I (AvaII) (10 U/µL)

Protocol tips

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	PCR products were submitted to enzymatic digestion with Eco47I (Fermentas, Lituania) in the following reaction conditions for a final volume of 15 µL: 1 unit of enzyme (0.1 µL), 1.5 µL of enzyme buffer, 7 µL of PCR product and sterile deionized water. Reactions were incubated at 37 °C for 12 h Digestion products were revealed by 2% agarose gel electrophoresis, subsequently stained with ethidium bromide and visualized using an UV transilluminator.

Protocol tips

PCR products were submitted to enzymatic digestion with Eco47I (Fermentas, Lituania) in the following reaction conditions for a final volume of 15 µL: 1 unit of enzyme (0.1 µL), 1.5 µL of enzyme buffer, 7 µL of PCR product and sterile deionized water. Reactions were incubated at 37 °C for 12 h Digestion products were revealed by 2% agarose gel electrophoresis, subsequently stained with ethidium bromide and visualized using an UV transilluminator.

Publication protocol

Genotyping was carried out through PCR amplification using specific primers (Forward: 5'-GTCATCTTCCTTGCCTGTTTAG-3'; Reverse: 5'-TTTCCACAAGGAGTTTTCAAGTT-3'), allowing amplification of a 228 bp within the sequence of the 13th exon. PCR amplification reactions were performed in a final volume of 25 µL containing 50 ng of genomic DNA, 200 nM of each primer, 0.2 mM of dNTPs (dATP, dTTP, dGTP, dCTP), 1 unit of Taq DNA polymerase, 2 mM of MgCl2, PCR buffer (KCl 50 mM, 2 mM MgCl2, Tris-HCl, pH 9.0) and sterile deionized water. The method was carried out in a Mycycler TM thermocycler (BioRad Laboratories Inc., Hercules, CA, USA) under the following temperatures scheme: initial denaturation at 98 °C for 3 min followed by 35 cycles consisting on 95 °C for 1 min (denaturation), 66 °C for 1 min (annealing) and 72 °C for 1 min (extension) with a final extension at 72 °C for 10 min. Amplification products were evaluated in a 2% agarose gel electrophoresis stained with ethidium bromide and visualized in an UV transilluminator. PCR products were submitted to enzymatic digestion with Eco47I (Fermentas, Lituania) in the following reaction conditions for a final volume of 15 µL: 1 unit of enzyme (0.1 µL), 1.5 µL of enzyme buffer, 7 µL of PCR product and sterile deionized water. Reactions were incubated at 37 °C for 12 h Digestion products were revealed by 2% agarose gel electrophoresis, subsequently stained with ethidium bromide and visualized using an UV transilluminator.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Eco47I (AvaII) (10 U/µL) below.

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