DNA Isolation - which acetate?

Hey, the most commonly used salt is sodium acetate (0.3M final conc, pH 5.2). This is routinely used in DNA precipitations. In solution, sodium acetate breaks up into Na+ and [CH3COO]-. The positively charged sodium ions neutralize the negative charge on the PO3- groups on the nucleic acids, making the molecule far less hydrophilic, and therefore much less soluble in water.

Other salts:

Use Sodium chloride (0,2M final conc) for DNA samples containing SDS since NaCl keeps SDS soluble in 70% ethanol so it won’t precipitate with the DNA.

Use Lithium Chloride (0.8M final conc) for RNA. This is because 2.5-3 volumes of ethanol should be used for RNA precipitation and LiCl is more soluble in ethanol than NaAc so will not precipitate, but beware – chloride ions will inhibit protein synthesis and DNA polymerase so LiCl is no good for RNA preps for in vitro translation or reverse transcription. In these cases, use NaAc.

Use Ammonium acetate (2M final conc) for the removal of dNTPs, but do not use for preparation of DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the enzyme.

Ok thank you so far.

But what happens to the SDS when I am using sodium acetate? Does the NaAc keep it soluble in ethanol like the NaCl does?

And what is the difference between KAc and NaAc in connection to DNA precipitations. K+ has the same charge as Na+ so is there
any reason why NaAc is more commonly used than KAc?

In a nutshell:

Acetate is negatively charged; the low pH contributes to charge positively the DNA. A combination of this plus high salt molarity enhances formation of aggregates of DNA and facilitates the pelleting procedure.

Potassium acetate is used to precipitate dodecyl sulfate (DS) and DS-bound proteins, allowing the removal of proteins from DNA. It is also used as a salt for the ethanol precipitation of DNA.

I think NaCl is more soluble in ethanol than NaAc so will not precipitate, but not 100% sure on this one.

Ok thanks, again!

But one last question on this topic regarding the use of KAc and NaAc:

Did I understand correctly that one can use KAc right before a DNA alcohol precipitation
to get rid of the SDS. And in opposite to the KAc the NaAc would just keep the
SDS in the ethanol phase during a ethanol precipitation?

So in practise I should use KAc if my DNA solution contains SDS,
If it don't, I should prefer  NaAc for the alcohol precipitation?

Yes, when used with ethanol, KAc behaves as a salt allowing for precipitation to occur. Because both NaAc and KAc are acidic salts (that act as buffers), they neutralize the solution and the alkaline conditions lead to denaturation of the DNA. So, either or can be used, but I've read that KAc is more effective than sodium acetate.

Yes, use KAc with SDS because it does an effective job at pulling out cell debris, SDS, SDS-protein complexes.

In ethanol precipitation, the positively charged K+ ions shield the negative charge on the phosphate backbone of the DNA. As a result, non-ionic, hydrophobic interactions take place which provoke aggregation of the DNA.

I'm having the same flipping problem! The protocol and my instructor said something along these lines.

SDS and the single stranded DNA have hydrophobic interactions, which causes precipitation. However, this requires a neutralization of pH. This is where potassium acetate comes in; potassium acetate is a neutralizing buffer, lowering the pH to the point that SDS can associate with the single stranded DNA. The potassium salt of dodecyl-sulfate is insoluble. So, the potassium cation associates with the extra SDS, precipitating out the SDS as well, reducing the amount of possible contaminants.

Hi

check out these links for detailed info but basically it looks like the NaOH and SDS in Sol'n I rupture the cells and denature the chromosomal and plasmid DNA. In Sol'n II the Potassium acetate neutralizes the NaOH assisting to reform the circular plasmid DNA and participates the SDS out of solution.

Hope this helps.
John

http://www.bio.net/bionet/mm/methods/2000-June/083524.html

http://www.osmania.ac.in/CPMB/Training/M10.htm

Thank you very much for the "osmania" website.
There I found a lot of useful manuals on laboratory methods with very very nice explanations of the principles.
I've already read certain books but none of them ever gave such good and logical explanation why I should use the
cerain chemicals!

THANKS!

I believe KAc is for circular and linear DNA whereas NaAc is only compatible for linear DNA. NaAc would likely destroy a plasmid, whcih is circular DNA.

Can you please give further explanations on why this should happen?
I can't really imagine the reason behind this theory.

are you familiar with that notion?

not particular sure, my friend told me that with a stellar explanation but I didn't recollect. Could it be due to the acidity ?

Could it be due to histone proteins? plasmids don't have histone proteins, so nothing really aggregates