Use step cycles, not hold segments. Temperature too low in some positions of thermal cycler Perform a set of control reactions to determine if certain positions in the thermal cycler give low yields. The top must be closed for correct heating and cooling. Inhibitor present Reduce the volume of sample in the reaction.
Ethanol precipitate to remove inhibitors.
Missing reaction component Check the reaction components and repeat the reaction. The reaction must be overlaid with high quality, nuclease-free light mineral oil. Do not use autoclaved mineral oil. Reaction tubes not autoclaved Autoclaving tubes eliminates contaminants that inhibit amplification. Poor primer design Make sure primers are not self-complementary or complementary to each other.