The DNA fragment was excise from the agarose gel with a clean, sharp scalped. The gel slice was weighed in a colorless tube and 3 volumes of Buffer QG was added to 1 volume of gel. The gel was incubated at 50°C until the gel slice has completely dissolve and to help dissolve better, the gel was mix by vortexing the tube every 2-3 min during incubation. After the gel have dissolved, 1 gel volume of isopropanol was added to the sample and it was mixed. The QIAquick spin column was placed in a provided 2 ml collection tube and to bind DNA, the sample was applied to the QIAquick column, and centrifuge for 1 min. The flow through was discarded, the QIAquick was placed back into the same collection tube. To wash, 0.75 ml of Buffer PE was added