The purification of a DNA fragment for microinjection is extremely important. This chapter describes a rapid and efficient technique for isolating specific DNA fragments from agarose gels run in Tris-acetate buffer, and was first described by Vogelstein and Gillespie (1). Agarose blocks containing the DNA fragment of interest are cut from gels and dissolved in Nal, a chaotropic salt that at concentrations of around AM is able to solubilize agarose. Glass beads are then added, which, in this concentration of Nal, efficiently bind to the released DNA fragments. RNA, proteins, and other impurities fail to bind to the glass fragments. Following a few washing cycles, the purified DNA is eluted from the glass into a low-salt buffer. This method produces DNA of sufficient purity for most applications. No further purification is needed for the DNA fragment to be subcloned, labeled using standard methods, or recleaved by restriction endonucleases. However, further purification is recommended if the DNA is to be microinjected into fertilized one-cell eggs. Passage through a Sephadex G-50 column previously equilibrated in MiTE removes all contaminating solutes and solvents (e.g., ethanol) that might be deleterious to the egg. Filtration through the 0.45-(Jm filter removes particulate matter that might block the microinjection pipet.
Glass bead isolation fulfills all the criteria to be the method of choice for the isolation of DNA fragments from gels:
1. It is rapid. A fragment can be isolated and ready for further processing within 2 h.
2. The method is efficient. Eighty percent recovery can be anticipated in most cases.
3. Its simplicity means that a large number of samples can be processed simultaneously.
4. The reagents used are relatively nonhazardous. Additionally the oxidation of the Nal (used to dissolve the agarose) to a purple-colored compound indicates the location any spillages!
5. The DNA fragment is extremely pure following processing. The quality is such that the DNA can be directly used in all manner of enzymatic reactions, and with little further purification, it is suitable for microinjection into fertilized one-cell eggs.
The biggest disadvantage of the technique is that the recovery of small (<500-800 bp) DNA fragments is not efficient. This can lead to substantial losses, although some will always be recovered.
Another possible disadvantage of the technique is that large (>15 kb) DNA fragments might potentially be broken during the wash cycles. If a large DNA fragment were to bind to two or more glass beads, then on washing and, hence, separation of the beads, the DNA strand might be broken. In practice, we have not found this to be a problem.
2. Materials (see Note 1)
1. Powdered glass flint, available from glass supply companies (e.g., Eagle Ceramics Inc., 12267 Wilkins Avenue, Rockville, MD 20852).
2. Autoclaved, filtered (0.2 pm) water.
3. Concentrated nitric acid.
4. 40X TAE buffer: 1,6M Tris-HCl, pH 8.0, 0.8M sodium acetate, and 0.04MEDTA.
5. Ethidium bromide (10 mg/mL) in sterile distilled water.
6. Long-wave (365 nM) UV light transilluminator.
7. 6M Nal: This is prepared by dissolving 90.8 g of Nal and 0.5 g of Na2S04 in water to a final vol of 100 mL. Filter through a 0.45-pm Nalgene filter, and then add a few Na2S04 crystals to the filtrate. The Na2S04 crystals do not dissolve properly, but serve to prevent the oxidation of the Nal. Store at 4°C protected from light.
8. Ethanol wash solution: 50% (v/v) ethanol, 0 1M NaCl, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA. Store at -20°C.
9. Sterile (autoclaved), filtered (0.2 pm) MiTE (Microinjection TE): 10 mM Tris-HCl, pH 7.4, and 0.2 mMEDTA (see Note 2).
10. Sephadex G-50 slurry (see Note 3): This is prepared by swelling G-50 powder in water followed by autoclaving.
11. 1-mL Sterile disposable syringes.
12. Baked glass wool: Bake at 250°C for at least 3 h to destroy all contaminating nucleases.
13. Millipore (Bedford, MA) filter type HV (catalog number SJHV004NS, autoclaved).
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