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===Purify the product from the gel=== *Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel. *Cut out the correct band (175-800 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly. [[File:ZhangLab_2 2011-08-30 15hr 04min.jpg|350px]] *Centrifuge at top speed > 13000 rcf for 2 min to sheer the gel, remove the 0.5 mL tube. *Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour *Spin down all tubes at 13,000rcf for 2min *In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.
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