CV1 transfection using Lipofectamine 2000 - (Aug/25/2008 )
I'm working with African green monkey CV-1 cells and it is hard to transfect them. I used Invitrogen pBudCE4.1 vectors to clone my genes and had problems with transfection. I tried Fermentas and Roche transfection kits. Then, I ordered Lipofectamine 2000. I know it is highly efficient. I optimized it with a vector coding GFP. But now, I have problems with my transfection using pBudCE4.1 vector. My transfected cells died in a day. I tried it again, but the result is the same. Anyone using Lipofectamine 2000, or working with CV1 cells to help me?
Thanks in advance,
It it quite common that transfected cells start to die off after 24 hrs with lipo2000. Whats your purpose of transfection? Is it for transient or stable transfection? If you only want to study gene expression and regulation, do your assay between 24-36 hrs. If you need to establish stable transfectants, you need to use suboptimal dosages of lipo2000 to reduce the toxicity. Hope this helps.
Thanks a lot. To begin with, transient tranfection is enough for me. I want to use these transfected cells to isolate RNA to use in q-PCR. Generally, I did RNA isolation 72 hours after transfection. Is it a problem to isolate them 24 hours after transfection?
But in near future, I'm planning to construct stable cell lines. Do you recommend me to use less lipofectamine reagent?
The expression of reporter gene as measured by enzymatic activity peaks between 32-48 hrs. The level of RNA would be ahead of that. Try 24-36 hr.