- 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.
- 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.
- Transfer slice to a pre-spun (12,000 x g for 20–30 seconds),
pre-weighed PLG 2 ml Light tube and determine the weight of the slice.
- Add a volume (in µl) 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 65°C for 5–10 minutes.
- 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.
- 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). >
- 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.
- 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).
- Centrifuge as in step 6 above and recover the aqueous phase to a
suitably sized microcentrifuge tube.
- Add 0.25 volume of 10 M Ammonium Acetate and 2.5 volume of 100% Ethanol
to the sample and mix well.
- 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
- Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) ÒMolecular
Cloning, A Laboratory ManualÓ, 2nd edition. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY. pp.6.30-6.31.
|