Hello everyone. Hope you'll are doing well. I have a question and would be really grateful for your responses. I actually isolated RNA from a cell line using Trizol- followed by DNAse I treatment and then Phenol chloroform extraction. I washed with 70% Ethanol twice. I have pretty decent concentrations (2000-3000 ng/ul) and 260/230 ratios are all above 2 but the 260/280 is between 1.71-1.81 with most samples having it at around 1.74. This is a little disappointing because I always had ratios between 1.8-2. I ran the samples on agarose gel and I think the bands look intact with 18S, 28S and lower bands visible. However the bands ratios don't look ideal. I'm attaching the picture below. I eventually want to use the RNA for Taqman assays and any suggestions would be highly welcome. Thank you.
A simple sodium acetate precipitation of the RNA will clean up your A260/230 ratios. Here is the method I use:
- Add ultra pure water make up RNA samples to 100ul in total
- Add 300ul 100% EtOH & 10ul 3M Sodium Acetate per sample
- Vortex + incubate overnight @ -20oC
- Centrifuge full speed @ 4oC for 30mins
- Pipette off supernatant.
- Add 500ul 70% EtOH + mix
- Centrifuge full speed @ 4oC for 15-20mins
- Pipette off supernatant & air dry for at least 15 mins [check that no EtOH remaining]
- Resuspend in 30ul or 12ul (depending on RNA concentration)
Give it a go, it works really well. Just remember the RNA pellets will be difficult to see (pellet paint can help).
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