Resuspended DNA in false buffer! - How to precipitate? (Nov/13/2006 )
I dissolved a DNA pellet (which I retrieved from a maxi prep) in 100mMTris-Cl pH8,5 Instead of 5mM Tris-Cl...
How should I proceed now? I have never precipitated my DNA from buffer...
And: now that there's too much salt in the solution- would you recommend diluting with water prior to precipitation?
no one ?
Maybe the answer is too obvious? And I should google?
I think you need reassurance.
So do this
Ethanol percipitate your DNA with 1/10 volume of 3M Sodium acetate and 3X volume of 100% Ethanol.
So if I have 100ul DNA solution
add 10ul 3M Sodium acetate
add 300ul 100% Ethanol
Spin down at maximum rpm for 12mins
Remove supernatant, and wash with 300ul 70% Ethanol
And resuspend in the correct buffer.
Note that you are adding enough Sodium acetate to cause the final concentration of NaAc salt to be 300mM. So no worries
Also note that for percipitating DNA, more salt is not a bad thing. It just means a more thorough clean up with the 70% Ethanol
Not enough salt on the other hand invites trouble, as the DNA won't percipitate.
@perneseblue: Thank you! But what i still haven't understood: After the ethanol precipitation I won't have removed the salts. There's still a lot of Tris and NaAc... You say it's no problem, but what if the DNA is to be used in salt concentration-sensitive procedures such as sequencing or transfection?
Sorry for not making this clear.
The salt is removed in the 70% ethanol washing steps. (Very Important)
You can wash your DNA as many times as you want in 70% ethanol. The 70% ethanol keeps the DNA in percipitate from. While the 30% water in the mix carries away the salt.
So it goes something like this,
*you have DNA pellet, at bottom of tube
*Add 1ml 70% ethanol. Vortex pellet (if vortexing is safe to do on your DNA), invert tube many times if vortexing is out of the question
*Spin down 7min at max rpm
*Remove 70% ethanol
repeat addition of 70% ethanol as many times as you desire. Usually once is enough, but if you are feeling worried do it 2 or 3 times.
*Once you have removed all the 70% ethanol. Spin the tube again at maximum rpm for 2 mins.
Then carefully remove any residue 70% ethanol
*Air dry for about 5mins at room temperature to evaporate any bit of ethanol. Of course this time would depend on how dry your lab is, and what temperature it is at. Don't over do this step though, especially if DNA from a maxiprep. The DNA become so dry it won't disolve.
*THen resuspend the DNA in your correct buffer. If the DNA is very clean it should literally dissapear into solution (even cold solution.) If not heat at 60 Celcius for 5min.