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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!

-Wolverena-

QUOTE (Wolverena @ Dec 2 2008, 05:53 PM)
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 have no idea, just a suggestion: Change (make new/borrow) 3M NaAcetate solution.

-cellcounter-

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.

-Cheamps-

QUOTE (Cheamps @ Dec 5 2008, 01:55 PM)
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!

-Wolverena-

QUOTE (cellcounter @ Dec 2 2008, 11:11 PM)
I have no idea, just a suggestion: Change (make new/borrow) 3M NaAcetate solution.


I changed the buffer....same result. Thanks for the suggestion, though!

-Wolverena-

QUOTE (Wolverena @ Dec 5 2008, 01:10 PM)
QUOTE (Cheamps @ Dec 5 2008, 01:55 PM)
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!



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

-Sheri-

QUOTE (Sheri @ Dec 5 2008, 10:00 PM)
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!

-Wolverena-