DNA for microinjection can be prepared from the plasmid or the cosmid clones by any of the standard techniques of lysozyme/Triton X-100 lysis or lysozyme/alkaline lysis followed by banding of supercoiled molecules in ethidium bromide/CsCl gradients. The many commercially available plasmid preparation kits, using alkaline lysis followed by anion-exchange column purification of DNA, provide DNA of a suitable quality with the minimum of time and effort.
Vector-free DNA is isolated by restriction enzyme digestion followed by preparative agarose-gel electrophoresis. This latter step is very important. DNA fragments for microinjection must be free from all contaminants that may be toxic to the eggs and all particulate matter that could clog up the injection pipet. Isolation of DNA from agarose gels by binding to glass beads (19) followed by passage through a Sephadex G- 50 column and filtration though a 0.45-|m filter provides such DNA. The method is described in the following protocol. However, glass bead DNA isolation kits are also commercially available (e.g., Geneclean from Bio101, Vista, CA).
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