Figure 3.

Recombination by φC31 integrase between attP and attB sites in vitro. (a) Restriction maps of plasmids used and expected recombinant products. Detection of the recombination products in b–d was by restriction with EcoRI followed by agarose gel electrophoresis. Parental and recombinant products are indicated. (b) A time course of recombination in vitro. (c) No effect on recombination by MgCl₂ and abolition of activity by the S12F mutation. Recombination reactions were incubated at 30°C for 16 hr before analysis. The smear of degraded DNA in the presence of the S12F mutant integrase is probably because of contaminating nucleases. (d) Recombination between linear substrates. Linearized pHS20 and pHS23 were prepared by cleaving with ScaI, and the fragments were purified before use as substrates in in vitro recombination. Recombination reactions were incubated for 16 hr before analysis. Molecular weight markers (M) are as in Fig. 2.