Hi, everyone.
I was trying to extract total RNA from a 18 h LB culture from E coli. DH5a using the commonly used TRI Reagent (from SIGMA-ALDRICH, catalog no. T9424) and 1-Bromo-3-chloropropane. I followed the protocol in the provided manual and got quite good results from other E coli. samples. However, the protocol seems do not work with E coli. DH5a (only this strain) because I did not see any RNA pellets after adding 2-propanol to the aqueous phase and the 4 oC centrifugation. At first, I thought it was the cell numbers. But when I use more cells, no pellet at all again. There's no contaminants in any of the reagents I used here which I am sure of it.
I'm wondering if anyone can give me some advice or other handy method for DH5a. I want to see the charging level of a specific tRNA in the following northern blot. So
Actually, it's not a good idea to extract RNA from stationary phase (and 18 hours in LB at 37oC definitely corresponds to stationary phase for DH5alpha). The RNA yields are low, and the quality is quite bad. Still, you should be able to have some RNA in the end. There is nothing special about DH5alpha strain with respect to RNA content. It might have higher RNase background but if you use Trizol it doesn't matter too much. I used to isolate RNA from this strain pretty often, and it worked fine.
Could it be the RNA precipitate was thinly distributed along the wall and overlooked? Or maybe the aqueous phase was not acidic enough prior adding isopropanol? You may try adding 3 M Sodium Acetate (pH 5) to the aqueous phase, one tenth of volume, and then 2.5 volumes of isopropanol, and keep the tube at (-20)oC for about 2-3 hours prior centrifugation. Usually, there is no need to do that but it might help to bring RNA down if the total amount is low.
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