I recently joined a lab and was handed a protocol and cells for transfection using fugene 6. I prepared the plasmid DNA and the GFP signal was good the first time. However ever since then, I have not been able to transfect the cells again. I ran the plasmid DNA on gel and I get multiple bands according to the enzymes I used. My cells are not dead so the DNA is not killing them either. One potential issue that I see is that these cells were passaged numerous times. Also, I think I passaged them too thin once and it took a long time for them to recover (newbie to these cells). Do you think these two potential issues could be the problem?
I will appreciate your response. Thanks
It might not be the problem but make sure you vortex the fugene just before use and mix immediately after adding to the transfection medium.
Also try a fresh bottle of reagent, unopened vials can be stored for much longer at -20C.
Where possible always get the lowest passage number cells you can and make your own stocks. You can sometimes get wildly different results depending on the age of the cells.
Good luck!
It is normal for plasmid DNA detecting 2-3 bands. I have used Fugene 6 for lots of times and never face any problem. I put the Fugene 6 kit at 4 C for 1-2 years and it still works. I think the cells may have problem. You may take a fresh vial of cells and try it again.
Hi Sehrish, I would put my money on cells being the cause of your failure. Just get a fresh vial of cells and hopefully it will solve the problem.
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