FIG. 5.

Principle of discriminating dilution series. A mixed 6 μl sample, with all four matching overlap DNA fragments (A), is portioned into two 3 μl tubes (B). From the two aliquots, half of the material (1.5 μl) is taken to be supplemented with a Gibson-mix of 1.5 μl) (C). To each half, a set of primers is added: in the first case, the primers for full assembly; in the second case, primers for the first two fragments to be ligated; in the third case, again the primers for the full assembly but without Gibson mix; and in the fourth case, primers for the final two fragments to be assembled without Gibson mix (D). The remaining lower tube at (B) is diluted with 3 μl of water and taken as the input for the next dilution round.