Life Technologies

62 Life Technologies ™ | Limited Dilution Cloning Always strain cells before diluting for limited dilution cloning, whether or not there is clumping observed. If not using peripheral wells of a 96-well plate for cloning, add 200 µL/ well of sterile PBS to help minimize evaporation during incubation. Ensure that pools are ≥95% viable before seeding for limited dilution cloning. Cloning medium: • Use freshly thawed glutamine for cloning medium preparation. • Use unopened bottles of CD FortiCHO ™ Medium. • Prepare cloning medium (CD FortiCHO ™ Medium supplemented with 6 mM L-glutamine) fresh (within 1 day of intended use). Ensure you have sufficient time to complete the limited dilution cloning workflow once cell dilutions have been made. In other words, there are no stopping points once you have started limited dilution cloning. Mix cells gently after each dilution by inverting the capped tube 5–6 times; avoid foaming. Manually count the last dilution (1,000 cells/mL), and adjust the volume to add to cloning medium accordingly; for example: • Pipette 8 µL of the cell suspension into 12 wells of a 24-well plate (96 µL total volume). Allow the cells to settle before examining them under the microscope; no staining is required. Cells tend to migrate towards the edges of the well due to the limited volume per well, which facilitates counting. • If the cells are at 1,000 cells/mL, then you will count 96 cells. Limited Dilution Cloning Do:

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