Found 1 discussion for this experiment
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?
Found 2 matching solutions for this experiment
|To ensure reliable and reproducable gene expression, minimal media is recommended, though complex media also work with this kit.|
|To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).|
|Include DNAse treatment for 15-20min.
Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C.
Use water to elute the RNA that is warmed to ~60`C
RNA protect only stabilizes the RNA and lyses the bacteria, for further purification RNeasy mini or midi kits are recommended by the manufacturer.