Problem	Solution
Step 6 Cells are not suspended and have settled to the bottom of the EHS gel.	If the cell-EHS mixture in Step 5 is diluted with medium before setting, the cells may settle to the bottom of the gel. Be sure to aspirate the majority of the supernatant to avoid this problem.
Step 18 Excess EHS causes immunostaining background.	Colonies fully extracted from the EHS gel as in Step 8 Option A should have little or no background. Partial extraction of colonies by Step 8 Option B may result in background if EHS is not sufficiently broken down, and a haze or cloud of EHS adheres to cells. To avoid this, increase both the volume of PBS-EDTA used and increase the incubation time in Step 8, Option A (ii).