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2 years ago
2 years ago by Paul G. Macon
Hello! I used Trizol to extract total RNA from in-vitro cultured bacteria (1 X 10^8 cells). After phase separation, I mixed ~0.4 ml of the upper phase which contains RNA with 0.5 mL cold isopropanol. However, the amount of RNA when measured in Nanodrop was very low. In addition, the ratio between 260 and 230 was around 0.1 to 0.5. Is there a chance that my sample was contaminated by the Trizol reagent? When I collected the aqueous phase I made sure to not touch the lower phase. What should I do?
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|Helicobacter pylori strains (Table Table11) were grown with gentle agitation (120 rpm) in 30 ml of Brucella broth at 37°C until mid-exponential phase (OD = 0.7). For heat-shock treatment, the wild type (WT) culture was split into 15 ml-aliquots and one sample was subjected to heat-shock at 42°C for 30 min (heat-shock sample, HS). A volume of 10 ml cell culture was then added to 1.25 ml of ice-cold EtOH-phenol stop solution (5% acid phenol, in EtOH) to stop growth and prevent RNA degradation. Cells were pelleted, stored at −80°C, and then used to extract total RNA with TRI-reagent (Sigma-Aldrich), according to manufacturer’s protocol.|