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1 year ago
1 year 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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|Genomic DNA and total RNA were isolated from S. pneumoniae strains using a Wizard genomic DNA isolation kit and SV total RNA isolation system (Promega), respectively, following the manufacturer's instructions, except that cells were incubated with 0.1% deoxycholic acid (Sigma) at 37°C for 10 min before extraction. RNasin (0.5%; Promega) was added to extracted RNA to prevent it from degradation. cDNA was derived and amplified from RNA using an Access reverse transcription-PCR (RT-PCR) system (Promega) and target-specific primers.|
|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"