Uneven colony sizes (large mixed with small) (Colony Formation Assay)

Question

Accepted Answer

Some colonies are very large while others are tiny — suggests cells were not properly singularized at seeding. Common causes: 1) Cells were not pipetted into a true single-cell suspension before plating 2) Plate was disturbed shortly after seeding (cells clumped) 3) Cell state was inconsistent across the suspension Solutions: 1) Pipette suspension vigorously to break up clumps; pass through a 40 µm cell strainer if necessary 2) After seeding, place the dish gently in the incubator and DO NOT disturb for 24 h 3) Use cells in log-phase with uniform morphology