Help with Phenol/Chloroform extraction - (May/23/2007 )
I have been doing in vitro RNA processing reactions, and want to clean up the protein from the RNA before I load on a gel. After the processing reaction, I add acidic phenol:chloroform pH 4.3, remove the aqueous layer, add chloroform, remove the aqueous layer, and ethanol precipitate. I add glycogen before the ethanol precipitation. However, I am getting very, very low recovery (<10%). The species I am trying to recover are very small (<60 nt). Can anyone suggest what I might be doing wrong?? I think I am losing most yield during the extraction phases. The RNA I have is P32 labeled, and I always notice that there is more radioactivity in the organic phases than in the aqueous phase (at both steps). There is hardly any radioactivity in the sup following EtOH precipitation (everythign is in the pellet).
Thanks so much.
ok i think you should proteinase K treat your RNA. I typically add 4µl of 20mg/ml to 100µl reaction. 10' at 37°. Add then 104µl of 0.6M NaAcetate pH 5.5 solution (1vol) and glycogen, mix and THEN add 200µl of phenol.
spin and pipett the upper phase. Put it in 1ml EtOH 100% and let sit overnight at -20°. -80° should be better but i'm not sure that it would not freeze. Test it.
The day after spin 45' 20 000g
wash, dry, resuspend, denature 10' at 56° and quantitate.
Try your RNA extraction only with chloroform (one or two extraction) and proceed as you do usually.