To determine the optimal concentration of IPTG for producing RNAi phenotypes, we titrated the IPTG concentration from 10 mM to 1 pM plus no IPTG. We found that 1 mM IPTG gave us the highest penetrance of phenotypes (Table 2). In addition, we tested the effect of seeded bacterial density and growth phase on the ability to generate phenotypes; for both par-1 and unc-22, saturated cultures produced phenotypes as well as log-phase bacteria (see the Materials and methods section). We also compared feeding at 15°C versus 22°C and found that although there is some gene-specific variation in RNAi effectiveness between these two temperatures, there is no generalizable difference (data not shown).
