Hi everyone!
I am trying to knock-out a gene in breast cancer cell lines with CRISPR/Cas9 technology. I have used commercial CRISPR/Cas9 system from SantaCruz. SantaCruz's: CRISPR/Cas9 KO plasmids that contain GFP.
I sorted the GFP+ cells by FACS 24, 48 or 72 hours after transfection and performed a complete phenotypic and genotypic analysis after isolation of single cell colonies to confirm complete allelic knockouts but I don't have any knockout clones (I analysed more than 300 clones) even when I have high efficiency in the transfection.
Do you have any idea of what could be wrong?
Thank you!
Hi Nuria,
I am wondering if you have used only one gRNA or multiple gRNAs for your gene of interest! We have found that using multiple gRNA significantly improved editing efficiency.
Hi Nuria,
I am working on this plasmid too with breast cancer cell lines. Did you find out what was the problem? How many mg of plasmid and ul of transfection reagents did you use for each well (6 well plate)?
Hello Olena,
Unfortunately I haven't find out the problem yet. I use 2 ug of total plasmid for each well. I dilute the 2 ug in 100 ul of serum free medium and 8 ul of PolyJet in 100 ul of the same medium. Then, I mix the two dilutions and after a 15 minutes incubation I add the 200 ul to each well (which has pre-warmed fresh complete cell growth medium).
If you have low transfection efficiency, you can use tripsine and resuspend the cell pellet with the previous 200 ul and then transfer it to a well with fresh complete medium.
Hope you find this useful
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