contamination in RNA extraction - (May/10/2007 )
I know it's not very new topic but...I had RNA extraction with low 260/280 ratio, I was surprised because I used the same method and reagents the day before and I had no problems. The only things that probably changed was the time of drying of RNA pellets before I dissolved in formamide.
So I left my RNAs at 60°C for 10min and at 4°C O/N to let dissolve properly but the day after ratio was low again, and RNA concentration was the same (not low!!!).
I controlled all of them on agarose gel: they are good and consistent with nanodrop quantification.
Do you think I really have protein contamination?
and if I have, there is a protocol for re-extract them (I used TRIzol)?
thank you all
When you took your OD260/280 readings, did you dilute the RNA in water or TE? That can make a big difference with RNA. Check the troubleshooting section in the protocol that comes with TRIzol.
I don't dilute it at all, I can read it directly in formamide using nanodrop. Usually it gives right OD260/280 and concentrations.
you can phenol/chlo your samples with phenol pH 4.7
aha, can you explain me precisely how? and I must put my RNAs in water before or I can leave them in formamide?