I have transfected L929 cells with polio virus in T-75 flask. I left it for 48 hours and now I collected the media which is 15ml DMEM. I want the best way to isolate the viral RNA from this media.
I was thinking, on such a large scale, no RNA extraction kit would help, so I thought of a sucrose cushion, or the centrifugal concentrator. The centrifugal concentrator is easier to use, but I am not sure how much I am going to lose of the virus in the media.
How big is your virus? The concentrators come in a variety of sizes.
Thanks A lot Maria. It is around 7.5kb RNA, make a protein equals to 2.6*10^6 dalton. I am not sure what would be the best size? Have you ever tried using any of these concentrators?
Many thanks
Yes I have and I found them very useful to separate proteins by size and to concentrate them as well. You might want to keep an eye on the level of liquid since your prep runs the risk of running dry if all the liquid goes through.
Here's a link with a manual for the Centriprep concentrators. Their lowest limit is 3,000 Da.
http://web.mnstate.edu/provost/CentriprepMan.pdf
I hope this helps,
Maria
Thanks very much Maria. I i found a centrifugal tube with Molecular Weight Cut Off value 50000D which is around 1/3 smaller than my virus, and i found smaller than that. Based on the fact that my virus is around 2.6*10^6 dalton, i am not sure which might be the best size to use?
Many thnaks
I have had bad luck with centrifugal concentrators. Depending on the type of membrane, a lot of stuff can stick. If you have a lot of virus - great, but If not I think you'll get better recovery using ultracentrifugation.
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