RNA isolation / purification Yeast - Pichia pastoris

Generally it has been difficult to isolate high-quality RNA from yeast because of problems disrupting the cells. Use of enzymes to disrupt cell wall can alter gene expression profiles. Therefore, physical disruption can result in high quality RNA for all downstream processing. Use of DNAse and proteinase K will remove traces of DNA contamination and proteins respectively.

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Found 3 matching solutions for this experiment

Protocol tips
- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
"- 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"
Protocol tips
Briefly, cell disruption was performed with 3×2 minute bursts, using a BioSpec Products Mini–Beadbeater–96 (Bartlesville, OK, USA) and purified RNA samples were flash frozen in liquid nitrogen before storage at –80°C.
TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.
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