In Vitro Packaging of λ and Cosmid DNA

Abstract

This chapter discusses the in vitro packaging of λ and cosmid deoxyribonucleic acid (DNA). The efficiency of cloning a desired DNA fragment in bacteriophage λ depends on a number of factors such as the availability of a useful λ vector, the probability of obtaining a DNA fragment in a possibly enriched form, the efficiency of conversion of the ligated hybrid DNA molecules to plaque-forming particles, and the availability of a suitable probe for the detection of the hybrid phage. The λ and cosmid DNA, as well as in vitro recombinants, are conveniently packaged using a combination of two lysates, each of which is defective in another step of λ morphogenesis. Empty precursor particles accumulate after induction in bacteria containing a prophage mutant in gene D. This mutation can be complemented by the addition of the missing D protein, most effectively by the supply of an induced λ E– lysogen. This mutation affects the main capsid protein; in its absence, all other head proteins are available in a soluble form. In the presence of adenosine triphosphate (ATP), spermidine, and putrescine, DNA is packaged, the head matures, and tails—components of both lysates—are attached. The result is a DNase-resistant infectious particle, which can be stored like any in vivo-produced phage. © 1979, Elsevier Inc. All right reserved.