I've been using phenol chloroform extractions on my Chromatin IPs, but after the ethanol precipitation I either get very tiny pellets, or a smear along the side of the tube. I think it is something to do with my reagents as I have done this side by side with a coworker using his reagents and it pellets fine. We thought we could solve it by regulating the pH of my Sodium Acetate to about 5.3, but that doesn't seem to have helped. I precipitate with 500 ul sample, 2ul glycogen, 44ul Sodium Acetate, and 1 ml 100% ethanol. Any advice would be greatly appreciated.
If you get different results (Tiny pellets or smear), then it sounds like you are not getting everything the same each time, at all.
A small pellet to me sounds like you are not extracting as much as you anticipate. A longer mixing phase could solve this issue.
Smear along the side sounds like it is not pure at all what you extract. Wait longer for the phases to seperate and equilibrate.
If it still doesn't work, but your co-worker can do it - then ask for it and follow his/her procedure, note down EVERYTHING, pipette tips sizes, where you obtain your water, how much you shake, really everything.
Also, inducing precipitation can give different results based on how you introduce your non-solvent. I would test a few methods if I were you and check if they give different results. Typically fast mixing gives smaller particles, so you can tune into what size particles you want.
I don't usually do these precipitations, but these are the things I would consider from what you wrote.
Hope it helps.
Thanks for your input. I have worked side by side with my coworker and attempt to do everything exactly the same, which is why it's so puzzling that it isn't working as well in my hands. The smearing issue seems to have been somewhat resolved by pH adjustments, and I would expect phase separation should be sufficient given I spin them for 5 minutes, but perhaps I'll give them a minute after the spin to settle completely. I will try mixing for longer this next round.
What's interesting is that the latest experiment worked well on paper (very good qPCR results), but the pellet was invisible. Perhaps I will also add more glycogen to make the pellet more visible.
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