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causing high background.About stimulus: perform a time course study by collecting samples at various time points.SEAPcan collect samples from a single cell culture Luciferasemust set up multiple cell cultures to collect at different time pointsLow transfection efficiency: poor quality DNA,PH, et al.Cell density keep constant。 5080% confluent at the time of transfectionCell in mid log phase growth。 Transfection efficiencies may decrease for cell lines have been passaged for too many generationstips of transfection efficienc