I want to generate knockout cell lines (of C2C12) with the crispr/cas method. Then use FACS to get single cells in a 96-wells plate. After cell sorting I want to expand and clone the single cells, but I don't have a protocol for single cell clonal expansion. Especially, because C2C12 cells are hard to culture as single cells (from what I know from literature). Can anyone help me or please give me some advice?
Kind regards,
Floris
C2C12 are already clonal population. if single cells are not surviving do sorting again after culture, if you find mixed population. we do this method for Sh rna mediated knockdown.
I had the same problem with some of my very sensitive cell lines. Sorting single cells into 96-well plates was either killing the cells either through the harsh sorting or just because they are single cells. What I recommend is to plate the cells at a very low density so single cells are far from each other, yet all are growing in the same plate. Check on them every day, and later colonies will grow from the single cells.
I did a single cell sort for C2C12 to use CrisprCas last year, and I can expand just follow the regular protocol. I used 5 plates of 96w to obtain 15 grown clons but my problem was that any clon were differenciated correctly... I'm gonne a sorter again next week , if you want to share experience with culture or Crisprcas let me know. Good luck¡¡
Thanks all! I know that C2C12, after a certain amount of passages, have trouble differentiating, but proliferation can be upregulated. I hope to apply CRISPR/Cas9 soon and share the experience!
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