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===Size Selection=== [[File:ZhangLab_2 2011-05-26 17hr 09min.jpg|400px]] *Prepare 3 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 (~200 nt in this case), transfer gel slice to shearing assemblies *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 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. *Spin down all tubes at 13,000rpm 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. *Perform ethanol precipitation *Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray {| border="1" align="center" | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume Needed''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- |100% Ethanol||align="right"|2.5X Sample Volume||align="right"|950uL |- |3M NaOAC||align="right"|0.1X Sample Volume||align="right"|38uL |- |Glycoblue||align="right"|1/300X Sample Volume||align="right"|2uL |- |Sample||align="right"|Sample Volume||align="right"|380uL |- |Total Volume |colspan="2" align="right"|1370uL |- |} *Mix all content by repeat pipetting using multichannel pipette *Store in -80C for more than 20 mins or forever *Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge *Centrifuge frozen 1.5mL tubes at 10000rpm for 30m at 4C *Remove all liquid '''CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE''' *Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube *Centrifuge at 10,000rpm for 5min at 4C *Remove all liquid, air dry sample for 5min or until samples dried out *Resuspend the DNA with 20uL RNAse free H2O
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