IGEM:Harvard/2006/GelPurification

Gel Purification
final concentration) preferably at longer wavelength 5 hours) the tube extinct.html| biopolymer calculator] to the desired level (e.g. 10 uM or 20 uM)
 * Pour a 8% acrylamide gel
 * Prepare 120 mL of gel solution
 * 57.6 g urea (8M urea final concentration, MW 60)
 * 24 mL 40% acrylamide stock (19:1 acrlylamide:bisacrylamide, 8%
 * 24 mL 5xTBE (1xTBE final concentration)
 * 700 uL 10% ammonium persulfate (APS)
 * 80 uL TEMED
 * Prepare gel comb, plate and spacer assembly
 * Pour gel into plate and spacer assembly
 * Let polymerize
 * Set up gel in gel electrophoresis apparatus, pour in 1xTBE
 * Resuspend oligos in 300 uL TE
 * Take 150 uL, add 150 uL 2xGLB (containing urea)
 * Incubate for 3 minutes in a ~50C water bath
 * Run at 180 volts for 1 hour
 * Disassemble plates
 * Place gel on plastic wrap
 * Place white sheet of paper underneath plastic wrap
 * Cut out bands illuminated by a handheld UV transilluminator,
 * Add 2 mL crush and soak buffer
 * Rotate on 37C shaker overnight
 * Recover supernatant into two x 1mL eppendorf tubes
 * Speedvac down to ~300 uL (approx.
 * If one speedvacs below 300 uL, then add water back to ~300 uL
 * Add 900 uL ethanol to each tube and invert six times gently
 * Incubate at -20C for 30 minutes
 * Spin 16100 rcf 20 minutes at 4 C
 * Decant supernatant
 * Spin tubes briefly in microfuge to collect liquid on the bottom of
 * Pipette out remaining liquid
 * Add 500 uL 75% ethanol, gently invert tube six times
 * Spin tubes 16100 rcf 2 minutes at 4 C
 * Remove the supernatant with pipettor
 * Repeat 75% ethanol wash
 * Speedvac tubes for five minutes
 * Add 100 uL TE to each tube
 * Incubate at room temperature for ten minutes
 * Combine the two 100 uL into 200 uL
 * Measure A260 and A280 for 4 uL DNA + 76 uL TE
 * Use 80 uL TE as a blank
 * Calculate concentration of DNA using [http://paris.chem.yale.edu/
 * Add 190 uL DNA + calculated volume of TE to bring the concentration