Protocols

Cloning cells by limiting dilution

Posted on Monday, June 07, 2004

Description
This protocol describes a method that can be used to clone individual cells. This is done by limiting dilution and examination of plates for single cells. Growth factors or selection components may be added to specifically amplify a particulat phenotype required.

Procedure
1) Adjust cell density to 1 x 106 cells /ml.

2) Dilute by 100: 200 ul cell suspension + 20 ml medium to give 1 x 104 cells/ml.

3) Concentration of cells is now 10,000 cells per ml. Therefore to get 50 cells in total = 5ul, 300 cells in total = 30 ul and to get 1000 cells = 100 ul.

4) Prepare 50 cells/10 ml, 300 cells/10ml and 1000 cells/ml. By adding 5ul, 30 ul and 100ul reciprocally to 10 ml of media.

5) Label 3 96 well plates wit 50, 300 and 1000. Aliquot 100 ul from each 10 ml cell suspension (50, 300 and 1000 cells) into each of the 96 wells in each labelled plate. This will give 0.5 cells/100 ul for the 50 cells suspension, 3 cells/100 ul for the 300 cells suspension and 10 cells/100 ul fo rthe 1000 cells suspension.

6) Leave at 37 degrees for 1-2 hours before examining each well for single cells. Mark those well with single cells.

7) The probablility of success is greater if fewer cells are inoculated onto the plate. Cells should not be transferred too early in order to prevent dilution of the cells. Cells should be transferred in sequence from the 96 well plate into a 48 well, 24 well, 12 well etc until it is possible to continue cultivation in flasks.

8) In order to guarantee colnability at least 2 cloning cycles should be carried out.

Recipes


Supplies


Tips


Submitted by: Lizzie