PCR procedure
The PCR master mix contained 12.5 µL dH2O, 2 µL 10x
buffer, 0.4 µL 10 mM dNTPs, 1 µL of each primer (10 mM), 2 µL 25 mM
MgCl2, and 0.1 µL 5 U/µL Taq polymerase (Invitrogen,
Carlsbad, CA, USA) per reaction. Reactions were run with an initial
denaturation step at 94 ˚C for 2 min, 40 cycles containing denaturation
at 94 ˚C for 30 s, annealing at 61 ˚C for 45 s, and extension at 72 ˚C
for 2 min, followed by a final extension at 72 ˚C for 7 min and a
cooling period at 10 ˚C for 2 min.
The PCR product was visualized on a 2% agarose gel by observing a 250
bp band. We extracted the 250 bp band with a High Pure PCR Product
Purification and Gel Extraction kit (Roche, Basel, Switzerland) to clean
the product for sequencing.