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===Gel electrophoresis and gel-cut=== *10uL 6X loading dye was added into each reaction (total volume will be 10+50=60uL). Mix by vortexing followed by quick spinning. *Load 30uL samples into wells of '''2% agarose gel made with 14-well comb''' using the long beak tips. *Run the gel with 135V for 25min. [[File:sam042310-PCR products in big volume.jpg|500px]] [[File:sam042310-PCR products in big volume-post cut.jpg|500px]] *The cut gel slice (~100uL)was placed in 1.5-mL tube. Add 300uL ADB solution into tubes to dissolve the agarose gel. *Incubat on 55C hotplate for 10min. Briefly vortexing to mix the dissolved gel. *Transfer the melted agarose mixture onto Zymo-spin column in a collection tube. Centrifuge at 10,000xg for 30sec. Discard the flow-through *Add 200uL wash buffer (added EtOH into wash buffer first). Centrifuge at 10,000xg for 30sec. Discard the flow-through. *Repeat the washing step again. *Add 10uL clean water on the filter. Incubate at RT for 1min. Transfet the filter on a clean 1.5-mL tubes. Centrifuge at 10,000xg for 1min to elute the DNA. *Nanodrop the eluted DNA.
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