Can transfect reagent be frozen? - (Sep/06/2005 )
1.Can transfection reagent be frozen? will it work normally (lipofectin in my case) after thawing?
2.I am using pSIREN (BD Biosciences) for RNAi. It has no marker for selection. Is it possible to make stable cell lines using this vector? How to achieve it?
Thank you!
hi
1 : not recommended but if it's just one (two at most) time reagent is not that affected.
2 : you have first to clone a selectve marker gene in it (easier way)
or 2 : after transfection : you'll have do do cell clones (hardest way because you probably have to screen more than 100 clones to get 1 positive, more if you are unlucky, less if you're quite very lucky.
Fred
Instead of cloning a selectable marker into your vector, you can also co-transfect a plasmid with the selectable marker. Just make sure that you transfect at least 10 times more of your plasmid of interest compared to the selection plasmid.
Thank you for reply!
Hi, Theo
I still have a question. How can the cells maintain my RNAi vector even if co-transfected with another vector containg a selectable marker? What is the theory? I recently shifted my model from yeast to animal cells so that I am not experienced about transfection. I wonder if the cells will lose my RNAi vector under no selection pressure. Could you explain more how plasmid w/o marker replicates along with cell proliferation? Thanks!
I still have a question. How can the cells maintain my RNAi vector even if co-transfected with another vector containg a selectable marker? What is the theory? I recently shifted my model from yeast to animal cells so that I am not experienced about transfection. I wonder if the cells will lose my RNAi vector under no selection pressure. Could you explain more how plasmid w/o marker replicates along with cell proliferation? Thanks!
I believe that co-transfection is as efficient as expression of your selectable marker from the same plasmid. For integration, the plasmid has to break somewhere. If you have it all on one plasmid, it might break just is your gene of interest, leaving you with a resistant cell lacking the desired expression of this gene. And similarly, co-transfection might also result in a cell containing only the selection marker.
Now, how to keep expression from your gene/siRNA? If your gene/siRNA is toxic, you will lose expression very rapidly anyhow. If expression is neutral, you might loose expression very slowly, even when the plasmid remains integrated. Only if the expressed gene/siRNA is required for survalval/cell growth or whatsoever, you can be sure you keep the expression very stable for a long period (for ever???)