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Star Republic: Guide for Biologists

Cloning by limiting dilution

The aim is to obtain clones derived from a single cell.

Protocol I

1. Prepare a suspension of cells in 10 ml medium that contains 1000 viable cells.

2. Prepare another suspension of cells in 10 ml that contains 100 viable cells.

3. Dispense 0.1 ml of the higher concentration (10 cell/0.1 ml) into 48 wells of the 96-well plate. Dispense 0.1 ml of the lower concentration (1 cells/0.1 ml) into the remaining 48 wells.

4. Incubate the plate for 4-7 days without changing the medium. Observe for growth of isolated clones.

Protocol II

1. Prepare 8 tubes. Dilute cells in 2.5 ml of medium at the concentrations of 20, 10, 5, 2.5, 1.25, 0.6, 0.3, and 0.15 cells/ml.

2. Use two 96-well plates. Dispense 0.1 ml of each dilution into 3 columns (24 wells).

3. Incubate the plate for 4-7 days without changing the medium. Observe for growth of isolated clones.