IGEM:Harvard/2006/GelPurification

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Gel Purification

  • 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%

final concentration)

      • 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,

preferably at longer wavelength

  • 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.

5 hours)

    • 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

the tube

  • 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/

extinct.html| biopolymer calculator]

  • Add 190 uL DNA + calculated volume of TE to bring the concentration

to the desired level (e.g. 10 uM or 20 uM)

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