The enzymatic amplification was performed in a
thermocycler (M.J. Research, Inc.), programmed for 40 cycles.
Each cycle was composed of one denaturation step at 94 ºC
for 15 sec; one annealing step at 35 ºC for 30 sec and one
extension step at 72 ºC for 1 min. After 40 cycles an extra
extension step was performed for 7 min at 72 ºC.
Amplification products were separated by gel electrophoresis
on 1.4% agarose gel in TBE buffer (90 mM Tris-borate and
2 mM EDTA) containing 10 mg/ml of ethidium bromide.
DNA bands were photographed under ultra violet light,
utilizing the photo documentation system, Eagle Eye II
(Stratagene).