Exosome isolation problems

2 posts / 0 new
Last post
jseibert
jseibert's picture
Exosome isolation problems

  Hello All,

I just started an exosome isolation (from plasma) protocol (by invitrogen) this week and I am having trouble pelleting the cell debris. I first thaw all my samples on ice, then stick them in the centrifuge at 2000 x g for 20 min @ RT. After this step, there is somewhat of a pellet at the bottom (I place all of my microcentrifuge cap hinges on the outside so even if I can’t see the pellet, I still treat it as though there is a pellet). As I am aspirating the supernatant (using either a p1000, p200, or p100) I seem to pick up cell debris right around where the meniscus is located (the debris also comes from the same side of the tube that the pellet is located on). I transferred the supernatant anyway to a new tube because the protocol calls for another centrifugation step at 10,000 x g for 20 min. However, the same problem came up, so I spun it down again at 15,000 x g for 20 min. It still did not fix the problem.

Is there anything I can do to not pick up any cell debris? I would like to refrain from using a smaller pipeteman (less suction) because this would take more time.
Any ideas would be greatly appreciated!
Jake

Chin Fen Teo
Chin Fen Teo's picture
Hi jseibert,

Hi jseibert,

My experience is
(1) to pipet VERY slow/gently;
(2) not to let the samples sit "idly" after the centrifugation (i.e. process quickily right after the centrifuge stops);
(3) yes you are right, use the smaller pipet...

Good luck!