IGEM:Harvard/2006/GelPurification

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)