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Products were further gel purified by using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and quantified by comparison, in 1% agarose gel, with Low DNA Mass Ladder (Invitrogen).
The gels were digitally imaged using a UVP Bio Doc-It camera (Ultra-Violet Laboratory Products, Upland, CA, USA), and the concentration of each PCR product was determined by comparing the intensity of each band to a standard curve derived from a low DNA mass ladder (Invitrogen Corp.