I've never used a speedvac, but it was suggested to me in order to concentrate my RNA. I was told I need to know which temp and time (and I can't remember what else) to set up as my parameters. My sample volume won't be any more than ~35ul.
Any suggestions?
Thats depends of the kind of speedback machine, I mean, the model.
In my experience, In a old speedback, at medium speed, took me approx. 40 minutes to concentrate from 60 uL to 10 uL at room temperature without problems to the RNA.
Godspeed!
An ethanol ppt of RNA can dry completely under a fume hood (with adequate pure air-flow) in about 2 hours.
But, you will find that it goes back into solution (with nuclease-free water) stubbornly after that. After drying for 2 hours, add your desired amount of nuclease free water, vortex, microfuge, vortex, microfuge, vortex, microfuge (each time for 10 seconds), then, heat for 2 minutes at 60C, then vortex, microfuge, vortex, microfuge, vortex, microfuge (each time for 10 seconds), add an appropriate amount of RNAseOUT, vortex, microfuge, then store at 4C for as long as you like. This concentrated and protected state of the RNA is good for 3+ years. I highly suggest against freezing it (e.g., do not freeze-thaw it ever). This is contrary to popular opinion, but, if you know anything about "RNAScope", and how tough RNA actually is (under the right conditions), you will know what I am talking about.
Always keep in mind that by concentrating your RNA via speedvac also will concentrate any other compounds present in your sample. If RNA quality is fine (ratio 260/280 and 260/230) I have good experiences using the speed vac. Otherwise I strongly would recommend precipitation. The settings totally depend on different factors: kind of machine (centrifuge-like with different temperature settings or old-fashioned one with separate cooling trap), volume and concentration of RNA of the sample. I always did the procedure step-wise (about 10 min and always marked the volume left on the outside of the tube).
If your sample is really RNase-free there is totally no problem to store it for long time at 4°C as recommended by Jack. But if not, than -80°C is better, but avoid cycles of freezing and thawing, better freeze aliquots.
Good luck!
The speedvac that I would be using is a ThermoFisher Savant SPD1010.
I was warned about using a cleanup kit and/or precipitation, as there is a chance of RNA loss. My concentration is already low as it is, so that is a concern.
I came across RNAstable tubes that can be used to concentrate samples. Their protocol states using no heat, for 1hr, for my volume (20-50ul sample). I tried doing a test run with regular tubes and water. There was no much drying (if at all) after 1 hr. I'm not sure if it's because I wasn't using the RNAstable tubes, but following their parameters. One parameter I didn't change (because it wasn't mentioned in the protocol) was the vacuum pressure. I left it with the default, which is 30. Not sure if that plays a role.
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