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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 4 matching solutions for this experiment
|The total RNA was isolated using the illustra RNAspin kit™ (Ge Healthcare, UK) and ribosomal RNA depletion was performed using the RiboMinus™ transcriptome Kits (TermoFisher Scientific), followed the manufacturer's recommendation. The integrity of RNA was checked in agarose gel (1.5%) in a denaturant condition and the amount of RNA was determined using a spectrophotometer (Figures S7, S8) (BioTek Sinergy HT).
|Pre-treatment with Lysozyme (40 ug/ml)|
|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
|Following RNA isolation from the P. aeruginosa and S. aureus co-culture, the samples were subjected to rRNA depletion using the MICROBExpress or Ribo-Zero kits. Bioanalyzer electropherogram traces revealed that, while rRNA was reduced upon treatment with the MICROBExpress kit, little to no traces of rRNA were detectable upon treatment with RiboZero|