I want to cotransfect CRISPR/CAS9 and donor plasmids, in Goat fetal fibroblast cells and epethelial cells using BTX EC2001, 630B cuvette chamber. But transfection effincy very poor. Kindly suggest me protocol for efficient transfection.
Transfection in fibroblasts is generaly not the easiest and certainly not high efficiency. Do you have the ability to try using Lipofectamin or any other chemical transfection reagent?
There are a handful of published protocols for electroporation and chemical transfection which I will link below, although each company/machine usually has its own suggestions and of course optimization of transfection is extremely cell specific. The protocol lists I link do not mention goat cells but there are similar cells for a jumping off point.
Electroporation- https://www.thermofisher.com/de/en/home/life-science/cell-culture/transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html
Lipofection- https://www.thermofisher.com/de/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html
Also which plasmids are you using, and do you have any selection markers on the plasmids to determine transfection efficiency?
Hi Thakur,
I agree with Adam, may be another chemical method of transfection would help you (for instance PEG). Probably you would have gone through "Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts". This seems promising and would help you out with an increase in the transfection frequencies.
Good Luck!
With fibroblasts we've noticed really low transfection rates, and they require higher concentration doses of reagent to get good transfection. Lipofection of primary fibroblasts has been effective in our case (see https://altogen.com/product/fibroblast-transfection-reagent-fibroblast-cells/). Electroporation may work to get donor plasmids into the cells, but usually these can be quite large and may require condensers to ease their way into cells.
- Badges
- Science method