I want to transfect D10Cas9 with 2 sgRNAs to make a nickase, so I need a double+ transfection. When I transfect TMD8 with the Neon electroporation system viability drops to
If you're having problems with cell viability in controls, your transfection conditions are likely to blame. Look into other protocols and reagents for lymphoma cell lines, and there are also CRISPR/Cas9 guides that could help you out as well (see https://altogen.com/product/ramos-electroporation-kit-lymphoma-cells-crl1596/ for lymphomas and https://altogen.com/product/cho-transfection-reagent-chinese-hamster-ovary-cells/ for a Cas9 protocol). Usually suspension lymphoma cell lines are electroporated, as you have done, but you might also want to look into chemical means of transfection.
Thank you Michael, I will have a look at these other reagents.
With controls I mean indeed only electroporation on this cell line. On other cell lines the protocol works fine, but my transfection conditions are bad for this specific cell line.
Hi Janneke,
I hope you have solved your problem. If not, have you tried the 24-well optimization protocol to find our the best condition for TMD8? Also, I found one paper using 1700 V, 20 ms, 1 pulse as the parameter to introduce Cas9 RNP to TMD8 cells. This paper can be found at https://www.cell.com/molecular-therapy-family/molecular-therapy/pdfExtended/S1525-0016(17)30571-3
Hope this help.
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