Isolation of DNA fragments for microinjection.

Abstract

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. Agarose blocks containing the DNA fragment of interest are cut from gels and dissolved in NaI, a chaotropic salt that at concentrations of around 4M is able to solubilize agarose. Glass beads are then added, which, in this concentration of NaI, 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-μm filter removes particulate matter that might block the microinjection pipet.