Hey all
I intend to isolate free circulating SiRNA in media using chloroform extraction. I have added chloroform to media containing SiRNA to get the phase separated. The Chloroform came in the lower layer. Why is this so? Even after vigorous vortexing the chloroform is still beneath the eppendorf tube instead of coming up.
Is this common? I had similar experience with trizol extraction s well. (only once though)
Kindly add your insights as to why this happened
Thanks and best
if you mix chloroform with water the chloroform being more dense (d=1.5) and not miscible with water it will form the lower phase; your result is OK. if you add enough salt to the water (cesium chloride for example) to have d>1.5 then the phase will reverse. for nucleic acid purification it is more convenient to have the aquaous phse on the top: after centrifugation take most of the aqueous phase transfert in another clean tube and precipitate the DNA/RNA by adding ethanol...
Bit late to respond here, but...are you just adding chloroform directly to the culture medium? That seems like a slightly odd thing to do.
As noted, chloroform is heavier than water (which culture media mostly is), and also does not mix with water, so...sinking to the bottom, leaving the media above, largely unaffected, is the expected outcome.
I would suggest you use a phenol/guanidium-based approach, like trizol: here the phenol WILL mix with the aqueous culture medium, and this plus the guanidium will lyse/denature/unfold anything unlucky enough to not be RNA. Do this, with enough trizol to make it work (i.e. 1ml trizol to 200ul media, or similar such ratios), and THEN initiate phase separation via chloroform. Phenol preferentially mixes with chloroform (which doesn't mix with water) so the mix separates out into distinct phases, and all your protein and DNA ends up in the phenol/chloroform phase (or at the interphase), and your RNA is left in the aqueous phase at the top.
So yeah: do that. Use trizol, and use lots of it. Ideally use the trizol/qiazol optimized for liquid samples (they make that).
- Badges
- Science topic
- Similar topics
- Medicine
- Physiology
- Biosignals
- Biomedical Signal Processing
- Statistical Analysis