Hi everyone!
I tried for the first time to do a gene Knockout in UMUC-3 cells.
I'm using the ready designed plasmids from Santa Cruz Biotechnologie company, same for the transfection reagent and the transfection medium.
The protocol provided suggests to use a concentration of plasmid going from 1 to 3µg/10µl and a vl of transfection reageant from 5 to 15µl. In total I tried 9 conditions with different cc and vls, after one day of seeding 2.10E5 cells in 6 well plate/3ml of medium , antibiotic free. I followed exactly the protocol provided by the company.
The problem is that after less than 24 hr of transfection, I noticed that the cells (both transfected with control and target gene plasmids) monolayer started detaching and the cells are floating in the medium, after 48hr the cell shape changed and they started giving signs of cell death.
Can anyone suggest any modifications please! as I did exactly as stated in the provided protocol.
I'm planning to:
- Perform a mock control, to test the toxicity of the transfection reageant on my cells.
- use a plasmid cc of 0.5µg/µl instead of 1, 2 and 3µg/µl.
I would appreciate any help or suggestion from you. Thank you.
Dear Nesrine Sassi ,
I think your cells may be experiencing toxicity from the transfection process. Here are some troubleshooting steps and adjustments that might help reduce cell stress and improve the transfection outcome:
Dear Muhammad Waqas ,
Thank you for your answer and for the provided explation.
1. The Mock control showed that the cells stay in a good shape until 72hr and do not detach from bottom. So we can exclude the toxicity of the transfection reagent as a possible cause.
2 and 3. The Plasmid concentraction was reduced to 0.5 µg for both control and target gene and it was tested with different volumes of TR. I faced the same problem were the cells died in less than 24 hrs. In this case I think it is still a problem of plasmid concentration since even the cells treated with the contol plasmid died.
Based on this I will directly opt for the 7th point.
Thank you again.
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