31 Cell Cloning
315
Subprotocol 7
Dilution Limit Method
Materials
- Centrifuge and centrifuge tube
- Pasteur pipette
- 96-Well culture plates
- Culture medium
- Cloning medium (see Comments of Subprotocoll)
Procedure
1. Select a culture and harvest cells by suspending the cells in the medium and by centrifuging the suspension at 150 g for 5 min.
2. Suspend the cell pellet in culture medium.
3. Mix a small amount of the cell suspension and cloning medium so as
to make a cell density of 100-200 cells/ml.
1. Distribute 10 fll of the cell suspension in cloning medium into each
well of a 96-well culture plate.
2. Examine each well, and select wells that contain a single cell.
1. Incubate the plate at the temperature used for culturing the original
cell population.
2. When a single cell divides, and the number of cells increases so as to
cover the bottom area of the well, transfer the cells to a larger vessel.
3. When the cells have multiplied to cover the whole bottom area of the
well, subculture the cells to a larger vessel. By repeating the subculture process in this manner, cells may be propagated to establish a
cloned population.
Comments
It is recommended that the whole procedure be repeated two to three
times.
Results
This method is suitable for density independent cells. Once growth of
isolated cells is obtained, subculture and recloning will be more easily
performed.
Equipment
Solutions
Dilution
Isolation of
a single cell
Maintenance
315
Subprotocol 7
Dilution Limit Method
Materials
- Centrifuge and centrifuge tube
- Pasteur pipette
- 96-Well culture plates
- Culture medium
- Cloning medium (see Comments of Subprotocoll)
Procedure
1. Select a culture and harvest cells by suspending the cells in the medium and by centrifuging the suspension at 150 g for 5 min.
2. Suspend the cell pellet in culture medium.
3. Mix a small amount of the cell suspension and cloning medium so as
to make a cell density of 100-200 cells/ml.
1. Distribute 10 fll of the cell suspension in cloning medium into each
well of a 96-well culture plate.
2. Examine each well, and select wells that contain a single cell.
1. Incubate the plate at the temperature used for culturing the original
cell population.
2. When a single cell divides, and the number of cells increases so as to
cover the bottom area of the well, transfer the cells to a larger vessel.
3. When the cells have multiplied to cover the whole bottom area of the
well, subculture the cells to a larger vessel. By repeating the subculture process in this manner, cells may be propagated to establish a
cloned population.
Comments
It is recommended that the whole procedure be repeated two to three
times.
Results
This method is suitable for density independent cells. Once growth of
isolated cells is obtained, subculture and recloning will be more easily
performed.
Equipment
Solutions
Dilution
Isolation of
a single cell
Maintenance