Tips for single cell cloning.
Tips for single cell cloning.
| If the customer is performing single cell cloning after the electroporation the cells need to recover 5-7 days before starting the single cell cloning. Some points to consider at single cell cloning in addition to the standard DEF-CS protocol: • At single cell cloning (1 well/96-well) DEF-CS GF-3 (diluted 1:1000) should be added at each medium change until the first passage is performed. Duration of DEF-CS GF-3 addition might vary depending on the growth characteristics of used cell line. • The time between seeding single cells and the first passage is longer than the normal passage interval since it will take some time to get the cells going, it can be up to approximately 14 days. The cells should be passaged when the cells are more or less confluent in the 96-well. • At this first passage, it is suitable to passage the cells from one 96-well using up to a 1:3 split ratio (based on area) i.e. from one 96-well to one 96- or 48-well. Centrifugation of the cell suspension should be avoided/is not necessary. • After the first passage the cells can be cultured in 96-well plates according to the user manual, using the recommended medium and seeding densities. The general troubleshooting guide can be used if needed. R&D are currently working on a protocol for single cell cloning but there is still some parameters that needs to be set. |