Figure 3.

Effect of Annealing of the 5′ and 3′ Fragments on Their Recombination

(A) Gel electrophoresis under nondenaturing conditions of a mixture of 10¹¹ molecules each of the [³²P]-labeled 5′ and 3′ fragments. Shown in the order of appearance: the mixture incubated for 40 min at 55°C in the presence of 0.5 M KCl; the same sample brought into a 1 mM Na-EDTA solution by passing through a Sephadex G-25 microcolumn; the desalted sample after heating for 2 min at 96°C; the mixture incubated for 40 min at 37°C in the presence of 0.5 M KCl. The last two lanes contain separate fragments.

(B) Colonies produced by the indicated number of molecules of each of the mixed 5′ and 3′ fragments that were annealed by heating to 96°C for 2 min in a Mg²⁺-free buffer containing 0.1 M KCl and then allowing the sample to cool down to 20°C during 1 hr (upper row), or preincubated in the same buffer at 20°C during 1 hr (lower row). Hybridization with the foreign extension-targeted oligonucleotide.