Fig 2.

Optimization of one-step SLIC. (A) Effect of the duration of T4 DNA polymerase treatment on one-step SLIC. One hundred nanograms of BamHI-digested pUC118-HMG (4.9 kb) was mixed with an insert (Ton_0709, 1 kb, 22 bp of homology) at a 1:2 vector-to-insert molar ratio in a 10-μl reaction mixture. The mixture was incubated with T4 DNA polymerase (0.6 U) at 22°C for the indicated times. The reaction mixture was than incubated on ice for 10 min, and competent TOP10 cells were transformed with the annealed DNA complex. (B) Effects of different vector DNA concentrations on one-step SLIC. Various concentrations of the vector were mixed with the insert at a 1:2 vector-to-insert molar ratio. The DNA complex was treated with T4 DNA polymerase (0.6 U) at 26°C for 2.5 min. After 10 min on ice, competent DH5α cells were transformed with the annealed DNA complex. (C) Effect of the vector-to-insert molar ratio on one-step SLIC. (D) Effect of the amount of T4 DNA polymerase on one-step SLIC. Each bar represents the mean ± the standard deviation of triplicates.