An improved method for recovering intact pulsed field gel purified DNA, of at least 1.6 megabases

The widespread use of yeast artificial chromosomes (YAC's) as cloning vectors, has created a demand for a reliable method to recover intact chromosomal DNA from preparative pulsed field gels. DNA isolated in this way can be used in a variety of procedures including micro-injection into mammalian cells (1) and the creation of cosmid and phage sublibraries (2). Schedl et al. (1) describe a procedure in which YAC DNA from a preparative pulsed field gel slice is concentrated into 4 % NuSieve and the intact DNA subsequently recovered by melting the agarose and digesting with Gelase. Here we describe a number of modifications to this method resulting in significant increases in the yield of DNA and speed of recovery. Intact DNA as large as 1.6 megabases was recovered from a pulsed field gel. This result was achieved primarily by improving the method for concentrating DNA and hence recovering it in a minimal volum