milky ethanol + viscous "pellet" - DNA precipitation (Dec/02/2008 )
Hi there,
I am trying to precipitate DNA after cell lysis, but there are some issues I can't explain. For cell lysis I am using SDS or CTAB or both, followed by phenol/chloroform/alcohol and chloroform/alcohol extraction, but I have some "problems" with the precipitation. After the extraction I am adding 0.1 volume 3M sodium acetate and 2 volumes 100% ice-cold ethanol. BUT, as soon as I add the ethanol the whole solution instantly turns milky (and stays like that). Why does it turn milky? The other confusing thing is that as soon as I add more salt (5M NaCl) it turns clear again. I am pretty sure that it is not sample because I also used a negative control (same procedure without the sample) that turned milky as well.
Any ideas what's happening??? ....I have no clue...
Cheers!
I am trying to precipitate DNA after cell lysis, but there are some issues I can't explain. For cell lysis I am using SDS or CTAB or both, followed by phenol/chloroform/alcohol and chloroform/alcohol extraction, but I have some "problems" with the precipitation. After the extraction I am adding 0.1 volume 3M sodium acetate and 2 volumes 100% ice-cold ethanol. BUT, as soon as I add the ethanol the whole solution instantly turns milky (and stays like that). Why does it turn milky? The other confusing thing is that as soon as I add more salt (5M NaCl) it turns clear again. I am pretty sure that it is not sample because I also used a negative control (same procedure without the sample) that turned milky as well.
Any ideas what's happening??? ....I have no clue...
Cheers!
I have no idea, just a suggestion: Change (make new/borrow) 3M NaAcetate solution.
Isn't the milky solution your DNA? I believe you can spin it out and wash it a few times and you should be good to go. I could be wrong on this, but normally after ethanol addition my solution turns milky as well and I take that to mean good DNA yield.
it would be awesome if all the milkiness would be DNA. Unfortunately, I think it's something else because I have problems with the "pelleting" as well. After centrifugation there is not "real" pellet. It's just a very viscous blob that moves when I try to take of the supernatant (like a viscous liquid). I have no clue what's happening or what I am doing wrong....it's just not the way it should be (shouldn't it be "very simple"?).
Cheers!
I changed the buffer....same result. Thanks for the suggestion, though!
it would be awesome if all the milkiness would be DNA. Unfortunately, I think it's something else because I have problems with the "pelleting" as well. After centrifugation there is not "real" pellet. It's just a very viscous blob that moves when I try to take of the supernatant (like a viscous liquid). I have no clue what's happening or what I am doing wrong....it's just not the way it should be (shouldn't it be "very simple"?).
Cheers!
maybe not spinning fast enough? We spin at 12000g for 12mins at 4 degrees and get gel-like pellets stuck to the eppy, that dont come off until the EtoH wash
Thanks for the reply everybody!
I am using a bench-top Eppendorf centrifuge at 14000rpm (can't say right now how many "g" that are) for 30min at 4C. I think it's something in the solution that makes it stay liquid.
So I tried to take off the alcohol, added TE and did another chloroform:alcohol extraction, followed by another precipitation....at that point there is a pellet. I am not sure how much DNA lost by doing this and if I can trust the result (the pellet)....Shouldn't it have worked the first time?
Cheers!