Pelleting tips and tricks

Описание: Pelleting (sedimentation centrifugation) separates particles based on their size and density. At low spin speeds, it can separate large suspended things and insoluble things from the liquid it was in (like if you spin down cells that were growing in suspension). And if you increase the speed you can start pull out smaller things that are suspended in the liquid (like separating precipitated DNA or RNA during an extraction). Go super fast and you can separate even smaller, well-suspended things like cellular organelles out of cell lysates (broken open cells) (nuclei, etc) (see theory note at bottom of post if you want to know more about why).

blog form: https://bit.ly/pelleting_practical

The stuff that gets pulled out collects as a solid pellet. And the stuff that doesn’t stays in the liquid portion, which we call the supernatant.

Sometimes what you want is in the pellet. Sometimes it’s in the supernatant. So be sure you know which you want!!! (And keep both until the end is unsure and/or if you need to troubleshoot because you didn’t find what you wanted in the part you kept).

Regardless of which you want, you need to isolate them after you separate them. Or else they can remix. Which defeats the purpose of centrifuging them! Sometimes this is harder than you’d think. But here are some tips to help make it easier.

First, you need to be able to find the pellet! If you have a huge glob of cell gunk, that’s easy to see. But if you have tiny pieces of DNA or RNA collected in a minuscule pellet, that makes things harder… thankfully, you should know where the pellet should be.

The pellet's position on the tube wall will depend on the tube angle during the spin, which dictates where the particles get pushed to. For fixed angle rotors, this is typically the “corner” on the tube bottom. For swinging bucket rotors, the tubes will swing out to horizontal during the run so the force will push those molecules to the center bottom of the tube.

When using a fixed angle rotor, orient tubes all in the same direction each time (I usually do it with the hinge straight out). This way you know where the pellet will be relative to the lid. Yo can also circle the pellet position when you remove tube. If you do washes and re-pelleting, orient the tubes the same way for
subsequent pelleting so that you don't lose yield from stuff being still stuck on other sides of walls.

Special note when pelleting small amounts of small things - you can often add a co-precipitant that will bind to the thing you want to pellet, thereby increasing its size and mass. Sometimes these co-precipitants are attached to dyes so you can see the pellet more easily as well. One such co-precipitate I use a lot is GlycoBlue which helps you pellet out (and see your pellet!) RNA and DNA. more about this here: https://bit.ly/na_precipitation; YouTube:    • DNA & RNA precipitation with alcohols & sa...  

methods of removing supernatant
these have fancy names but are simple*
*the challenge is not removing the pellet in the process!

decanting: simply pour off liquid
This works best when pellets are firm
with loose pellets you risk them getting poured out in the process.
It’s fast, so there's less risk of pellet resuspending but you need to be careful when you pour - so you collect only the supernatant and nothing more!
Being careful can mean leaving some liquid behind though in fear of losing pellet. So you may need to follow with aspirating to remove that remaining liquid (which can sometimes be super important, such as if it contains things that will interfere with later steps in your protocol).
aspirating: suck off liquid
Remove the liquid with a pipet or a pipet tip attached to a vacuum line (only if you don't want to keep the supernatant).
Be careful not to touch pellet with tip (especially with vacuum line!)
start with a bigger tool (e.g. P1000 for microcentrifuge tubes) to get as much off as you can, then switch to something with a finer tip (e.g. P200) to get into the crevices the fatter tips can’t reach so you get the last drops

oh no! where'd it go?!

Regardless of your separation method, be careful when separating supernatant from pellet so you don't disturb the pellet and cause it to "un-pellet".
Once spinning stops, remove supernatant ASAP
Where possible (e.g when there’s not someone waiting in line behind you to spin), remove tubes one at a time, and remove supernatant as soon as you remove the tube, while still holding tube at the angle it's been at.

If it’s the pellet you’re after, you often want to “resuspend” it in some fresh liquid. You typically do this by pipetting up and down lots. You may need to get a little more intense to break up firm pellets. This might involve vortexing (if your stuff’s not too sensitive), scraping the bottom of the tube against a tube rack, or sonicating briefly. Finished in comments