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Recovery of DNA from LMP Agarose1

Recovery of DNA from LMP Agarose1

  1. Resolve DNA fragments on a Low Melting Point (LMP) agarose gel in 1x Tris-Acetate-EDTA (TAE) buffer. Tris-Borate-EDTA (TBE) is not recommended as TBE gels are much more difficult to solubilize.
  2. Stain gel with Ethidium Bromide, visualize with a longwave UV light, and carefully cut out the band(s) of interest with a sharp razor blade. Caution: Wear gloves when handling Ethidium Bromide and stained gels.
  3. Transfer slice to a pre-spun (12,000 x g for 2030 seconds), pre-weighed PLG 2 ml Light tube and determine the weight of the slice.
  4. Add a volume (in l) of TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) equivalent to 5x the weight (in mg) of the slice and melt the LMP agarose in the TE at 65C for 510 minutes.
  5. Mix well to ensure LMP agarose slice is fully dissolved, allow the dissolved sample to come to room temperature, and then add an equal volume of room temperature Tris-buffer saturated Phenol (pH 8.0) to the sample and mix until homogeneous. Do not vortex.
  6. Centrifuge at full speed (12,000 x g or greater in a microcentrifuge) for 2 minutes to separate the phases. Note: If the resulting aqueous phase still appears cloudy, the extraction should be repeated with room temperature Tris-buffer saturated Phenol (pH 8.0). >
  7. Recover aqueous phase to a fresh PLG 2 ml Light tube and extract with an equivalent volume of room temperature Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1). Do not vortex.
  8. Centrifuge as in step 6 above . Recover aqueous phase to a fresh PLG 2 ml Light tube, and extract with an equivalent volume of room temperature Chloroform-Isoamyl Alcohol (CI, 24:1).
  9. Centrifuge as in step 6 above and recover the aqueous phase to a suitably sized microcentrifuge tube.
  10. Add 0.25 volume of 10 M Ammonium Acetate and 2.5 volume of 100% Ethanol to the sample and mix well.
  11. Incubate at room temperature for 20 minutes, pellet by centrifugation, wash pellet two to three times with cold 70% Ethanol, air-dry pellet, and resuspend in a suitable buffer.
References
  1. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp.6.30-6.31.

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