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==padlock probe library preparation== Step 7: Page Denaturing Gel 1. Divide the oligo mix into two tube. Add 40 uL Novex TBE-Urea sample buffer (2x) in each tube. 2. Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer. 3. Denature the DNA with loading dye on thermalcycler 75C for 15 min, then quickly transfer to ice, wait for 1 min. 4. Use a 2D well denaturing gel, add 0.5x TBE buffer. Flush the wells very well to remove all urea. 6.Pre-run the gel for 10 min at 200V, load the samples into the wells, run at 200V for 30 min. 7. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min. !!! Note: starting from this step, everything need to be done in the hood to avoid contamination. * Actually, two gel is OK for the oligo mix. Step 8: Purify the product from the gel 1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. <BR> Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. You need two assemblies for each PAGE gel. 2.Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half<BR> and transfer each half to one shearing assembly. 3. Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. 4. 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,<BR> shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. 5. In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm),<BR> and then transfer supernatant to 1.5mL tube. NOTE: Spin the Nanosep columns until only gel is left in there, because the gel will stuck the filter,<BR> and samples will deposit in the tube. Mix the gel and samples very well before spinning, or change to other column if necessary. [[Image:ZhangLab_2 2008-04-11 16hr 27min---1.jpeg]]<br> Note: the gel on the right was purified with MinElute purification kit before putting into gel. The bands in the original gel image was weaker and had less intensity compare to the gel without purification. This possibly suggests that purification will decrease with % yield.
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