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==2D Gel Size Selection== *I will use one 6% TBE-Urea gel 2D well to perform purification (one gel can handle 2-2.5ug) *Mix 40ul 2X TBE-Urea sample buffer in a 1:1 ration to make the final volume to 80ul {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" valign="bottom" | width="139" height="15" | Components | width="65" | 1 lane |- style="font-size:12pt" valign="bottom" | height="15" | 10bp DNA ladder | 1 ul |- style="font-size:12pt" valign="bottom" | height="15" | 2X TBE-Urea sample buffer | 5ul |- style="font-size:12pt" valign="bottom" | height="15" | H2O | 4ul |- style="font-size:12pt" valign="bottom" | height="15" | Total Volume | 10ul |} *Pre-run 6%TBE βurea (TBU) gel at 250 volts for 10-15 min in 0.5X TBE buffer. *Heat DNA and DNA ladder-loading dye mixture at 75 C for 10mins, quickly transfer to ice, wait for 1 min *load gel with ladder and probes *Run gel at 200V for 30 mins *Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min *Prepare 3X 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 *Cut out the correct band chop the band into small slices, split into 3x shearing assembly. *Centrifuge at top speed > 12000 rpm 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 50C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. *In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm) and then transfer supernatant to 1.5mL tube. [[File:ZhangLab_2 2014-01-10 15hr 20min.jpg|400px]]
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