? TROUBLESHOOTING 62Return the cells to the incubator and allow them to expand for 2–3 weeks. Add 100 μl of warm D10 medium 5 d after sorting. Change 100 μl of the medium every 3–5 d as necessary.63Inspect the colonies for “clonal” appearance 1 week after sorting: rounded colonies radiating from a central point. Mark off the wells that are empty or that may have been seeded more than a single cell.64When the cells are more than 60% confluent, prepare replica plates for passaging (one well for each clone) by adding 100 μl of D10 medium to each well in the replica plates. Dissociate the cells directly by pipetting up and down vigorously 20 times, and plate 20% of each of the resuspended volumes into the replica wells to keep the clonal lines. Change the medium every 2–3 d thereafter and passage accordingly.65Use the remaining 80% of cells for DNA isolation and genotyping (Steps 71–74).
