problem with RNA extraction using TRIZOL - (Sep/01/2006 )
Hi..I am using TRIZOL to extract the total RNA from cockroaches. After spinning down and following all the steps..I got the white pellet at the bottom which i don't think it's RNA pellet. Is it the protein? I wonder if i should make the extra step for spinning down to get rid of all the protein stuff. please help.
In danger of sounding both stupid and condescending at the same time, how do you know that the pellet is not RNA? If you have followed all the steps correctly, you should get RNA (mixed in with a bit of protein) at the end. The RNA pellet does look big.
Run some of the pellet on a gel. Or put it through a spectrometer. More information of what was actually done during the Trizol extraction would be a great help to ascertain what has gone wrong. I am not sure where you are in the Trizol extraction protocol when you mention "extra spin step"
May all your ligations be successful and your PCR products long.
I agree, try running some of the sample on the gel. The pellet you have is probably RNA and not protein. When using Trizol the RNA is in the aqueous layer, the DNA is in the interphase and the protein is in the organic layer. If you have good separation of those phases during the extraction you should get very little protein in your RNA and perhaps a little DNA. DNA is fairly easy to remove from RNA though so that isn't really a problem.