Escherichia coli recA− strains are usually used for cloning to prevent insert instability via RecA-dependent recombination. Here, we report that E. coli BW25113 (recA+) competent cells prepared by usi
The number of recombinant colonies for each transformation was counted. Restriction-ligation was performed with continuous incubation at 37°C or for 20–50 cycles (2 min 37°C+5 min16°C). Cloning for pR
All reaction mixtures contained 1 mM ATP and 5 Weiss unit T4 DNA ligase (Fermentas) and incubated at 37°C. Experiments were performed using 50 ng vector, and the corresponding amount of insert DNA at
