Flowchart of the self-activator preclearing step followed by forward two-hybrid screens. DB-X bait constructs are transformed and selected on synthetic plates lacking leucine (Sc-Leu). These plates are replica-plated (R.P.) onto two plates. One plate contains 20 mm 3AT (Sc+3AT#1) and is used to monitor the number of self activators prior to the preclearing step; the other contains 5FOA (0.2%) (Sc-Leu+5FOA) and is used to eliminate self activators. The Sc-Leu+5FOA plates are then replica plated onto Sc-Leu plates for recovery. The recovery plates are transferred to a second Sc+3AT plate (20 mm) (Sc+3AT#2) to assess the efficiency of self-activator elimination. If necessary, the whole procedure can be repeated. After efficient elimination of self activators, the plates can be used for forward two-hybrid screens. Representatives of the three plates indicated in red in this scheme are shown in the next two figures.
