How much salt can/should be used for DNA precipitation? - Is there a lower or upper limit? (Oct/08/2008 )

Hi there,

I have a question regarding the salt content for DNA precipitation. Does it matter how much salt is added (or not added) to precipitate DNA? Is there a lower or upper limit?

I have done cell lysis with a buffer containing CTAB and 1.4M NaCl, followed by Phenol-Chloroform-Isoamyl alcohol step and Chloroform-Isoamyl step. The next step is DNA precipitation. Is the NaCl that was added in previous steps efficient for precipitation or do I have to add sodium acetate to the solution? If I add sodium acetate, could it be that there is too much salt in the solution?

What is your opinion?

Cheers,
V

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There are such as things as too much and too little. Too much and it is hard to remove and thus affects down-stream applications, too little and DNA won't precipitate efficiently. The quantities required depends on the salt used for the extraction - ammonium acetate and sodium acetate are common ones, but NaCl, KCl and many others will also work, though it is best to avoid Mg salts as these are commonly used by DNases.

If the NaCl is at a final concentration of 1.4 M in your extraction, then it will work but you probably need to add a little more, up to about 2.5 M. I am not sure if the addition of different types of salt is additive or independent (e.g. if you were adding ammonium on top of the Na).

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Do you have access to the Molecular Cloning handbooks? I don't have them on hand at the moment, but there is a very nice table in the appendix that shows which salts can be used for ethanol precipitation, when it is preferable to use (or not use) each of them, and what the final concentrations should be in a precipitation reaction.

Here's the reference

Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes


Ginger.

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Hey guys,

Thanks for your replies. I appreciate it.

I checked the Sambrook. It says that the final concentration for NaCl is 0.2M. I guess I am way over that concentration. I am hoping that the higher amount of salt has no influence in the precipitation because it is required for CTAB lysis.

Cheers,
V

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You can always add water to reduce the salt concentration before doing your purification. That might prevent precipitation of too much salt with your DNA (i.e. more salt will stay in solution.)

Ginger

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just don't dilute it too much. You can't precipitate DNA with ethanol if it is too dilute. You will have to go through a silicon column or use butanol dehydration to concentrate the DNA.

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if there is too much salt, but can't dilute too much, would it be possible to do several 70% Ethanol washes (more than 2) to get out the salt?

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Sure. The table I referred to above also gives acceptable concentrations of nucleic acid for efficient precipitation using ethanol, isopropanol, etc., so you could also check it to see how much dilution would be too much.

Ginger

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Hi Ginger,

I found the section for concentrating nucleic acids (A8.12) in the Molecular Cloning Manual Volume 3 that describes the 4 different salts, but, unfortunately, I couldn't find the table you mentioned. So I am wondering if I am maybe looking at the wrong place. I really would like to see this table. Do you possibly have a copy of it or any idea where to look for it?

Cheers,
Verena

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