Transgene Release DNA Preparation

The transgene is released from vector sequences by restriction endonuclease (REN) digestion. Removal of prokaryotic sequences should be carried out as completely as possible (see Note 1). Depending on the materials available, several methods can be pursued to extract the linearized transgene from agarose gels: acid phenol extraction (see Subheading 3.1.2.), agarose treatment (see Note 6), and electroelution (see Subheading 3.1.3.). Electroelution is performed by trapping DNA onto a dialysis membrane. In practice, DNA is electroeluted from gel slices inside dialysis tubing or by using a system commercially marketed by ISCO. Since the latter is simpler to use, this approach is described in this subheading. For standard electroelution using dialysis tubing, we refer the reader elsewhere (38). For subsequent purification of DNA for microinjection, we usually use Elutip-D columns, with highly satisfactory and consistent results (see Subheading 3.1.4. and Note 7).

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