Electroelution

Posted on May 16th, 2016 by UNM CC

Materials

Electroelution gel box and elution block
0.25x TBE buffer
5 M KOAc with BPB (bromphenol blue)
100% EtOH
70% EtOH
T.E. buffer (10 mM Tris, 1 mM EDTA, pH 8)

Procedure

  1. Run your DNA fragment on a preparative agarose gel
  2. Add 350 mL of 0.25x TBE buffer into electroelution box. Buffer should cover the ports on the elution block but should not cover the top of the block. Remove any bubbles from the elution block by flushing the tubes with TBE buffer. Do not use higher concentrations of TBE buffer.
  3. Using a pipetman with a gel loading tip add 40 µL of 5M KOAc/BPB to the sloping tunnel of the device, starting at the bottom of the well, then gradually withdrawing the pipet tip as the well fills. The vertical tunnel should remain filled with TBE buffer.
  4. Cut gel fragment containing DNA band into 2 mm wide strips.
  5. Add 2-4 mm gel strips to the slot in the elution device, closest to the elution port.
  6. Electroelute at 100v. x50 min. for a 1 kb fragment.
  7. Using a gel loading tip remove 50 uL of the blue solution from the device.
  8. Let the solution settle and remove the remaining solution to bring the total volume to 100 µL.
  9. Precipitate the DNA by adding 200 µL of 100% EtOH. Incubate at -20ºC x30 min.
  10. Spin at 13,000 rpm, 4ºC.
  11. Wash 3x with ethanol:
    1. Add 1 mL 70% EtOH, mix gently.
    2. Spin x2 min. at max. speed. Decant off supernatant.
  12. Air dry.
  13. Resuspend in a minimum volume of T.E.
  14. Check concentration and quality of product by running 1 µL of DNA on a test gel with a DNA size standard.

Fero-Electroelution

Tags: Fero-DNA

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