The PCR2 reactions were carried out as follows: 98 °C for 2 min, then 10 cycles of (98 °C for 10 s, 65 °C for 20 s, and 72 °C for 30 s), followed by a final 72 °C extension for 2 min. PCR2 products were purified by electrophoresis with a 1.5% agarose gel and DNA concentration was measured by fluorometric quantification (Qubit, Thermo).
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