2 replies to this topic

[aquarella_7]

Hi! im performing a RNA purification with Trizol Reagent from peripherial blood mononuclear cells. During the process I had to precipitate the RNA with isopropanol overnight at -80ºC, and the samples appeared forezen at the other day,  someone told me that this could be the reason i saw degradation in some samples, is this possible??.
After that i have to precipitate the sample again to send them in dry ice (300 ul) with 2 volumes of absolute ethanol and 0,1 volumes of  2M sodium acetate pH 4. When i put the samples at -80ºC  i saw that they were frozen again! why this happened ? maybe the dilution of the ethanol was below the freezing point? i didnt want to freeze them because this can ensure more RNA degradation during the thawing cicles, could someone help me with this?? is there any other recipe for RNA precipitation that i could use?
Thanks a lot!

[Curtis]

last time I put my Iso Propanol in -80 but it didn't freeze.

What I guess is that you didn't add enough Iso Propanol. The more the better. your RNA won't dissolve in IsoP anyway and will precipitate, so don't be afraid if you put more IsoP.

Maybe after addition of Chloroform and collecting the top phase you had more volume compared to your IsoP?

and why do you need to keep your samples in IsoP in first place? We extract RNA, then add water with RNase Inhibitor, then keep in -80.

[aquarella_7]

HI!! I will try adding more ispropanol durig precipitation, but did you have any idea why the RNA (disolved in DEPC water) after washing it with 75% ethanol, might freeze in 3 volumes of 100% and 0.1 volumes of AcNa 2M (pH 4) at -80ºC???

Thanks!!