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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?
Found 3 matching solutions for this experiment
|For RNA to dissolve better and to yield high levels, preheat elution buffer at 95C.
Addition of β-mercaptoethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation.
To capture a higher amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.
Do vortex for 2-plus minutes. This will facilitate the removal of tightly bound proteins typically associated with RNA.
|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