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mistake during alcohol precipitation - (May/04/2008 )

Hi guys,

I had my RNA resuspended in 0.5% SDS which was not the way to go. So I tried to alcohol precipitate it. Unfortunately, I just found out that I should be using NaCL instead of sodium acetate!!
My RNA is now sitting in alcohol in -20C. What should I do to rectify the problem?

Should I just continue and then do a second precipitation with NaCl?

-calavera1984-

QUOTE (calavera1984 @ May 4 2008, 08:44 PM)
Hi guys,

I had my RNA resuspended in 0.5% SDS which was not the way to go. So I tried to alcohol precipitate it. Unfortunately, I just found out that I should be using NaCL instead of sodium acetate!!
My RNA is now sitting in alcohol in -20C. What should I do to rectify the problem?

Should I just continue and then do a second precipitation with NaCl?


I think you can continue and then do a second precipitation with NaCl.

-NTH-

I would guess that substituting sodium acetate for sodium chloride would not matter, but adding more NaCl will surely not prevent it from precipitating. Go forward and precipitate. Be sure to wash with 70% ethanol, perhaps more than once.

-phage434-

QUOTE (phage434 @ May 8 2008, 04:45 AM)
I would guess that substituting sodium acetate for sodium chloride would not matter, but adding more NaCl will surely not prevent it from precipitating. Go forward and precipitate. Be sure to wash with 70% ethanol, perhaps more than once.


Errr......I'm confused here. "Adding more NaCl will not prevent it from precipitating"??
So adding NaCl will still cause the SDS to precipitate in the presence of sodium acetate?

Anyway, I've continued the precipitation with sodium acetate and then did a second precipitation with NaCl instead. Samples seem to be fine. Thanks for the feedback guys.

-calavera1984-

DNA precipitates when the sodium ion concentration is high enough and when the alcohol concentration is high enough. Either sodium acetate or sodium chloride will work. You can precipitate the dodecyl sulfate by adding potassium (not sodium), since potassium dodecyl sulfate is insoluble. But you don't want to do that -- you want to get rid of the SDS.

-phage434-

QUOTE (phage434 @ May 9 2008, 05:44 PM)
DNA precipitates when the sodium ion concentration is high enough and when the alcohol concentration is high enough. Either sodium acetate or sodium chloride will work. You can precipitate the dodecyl sulfate by adding potassium (not sodium), since potassium dodecyl sulfate is insoluble. But you don't want to do that -- you want to get rid of the SDS.


Thanks. I'm learning a lot from these discussions.

-calavera1984-

Why exactly is sodium acetate used instead of sodium chloride any way? I always thought the high [H+] counteracts the P- on DNA and makes it drop out of solution? Is that the idea?

-killerkoz17-