Different lots or shipments of microplates may vary in the background fluorescence of the plastic. It is wise to check the background fluorescence levels of a new batch of plates by imaging an empty plate before running the assays.
If the β-gal expression is low, use a higher DDAOG concentration (e.g., 50 μM, or a 1:200 dilution of the DDAOG stock solution in Lysis and Reaction Buffer).
When the optimum incubation time has been determined, the enzymatic reaction can be stopped with stop buffer if desired (add NaCO3 to a final concentration of 0.33 M).
When pipetting, be careful not to introduce bubbles. Bubbles in the wells may refract light during imaging.