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Name: Jonathan
Status: Educator
Age: 30s
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Question:
With respect to precipitating DNA and RNA (maybe your answer is different for each) using salt and alcohol, what would be the optimum combination of salt type (LiCl, NaAcetate, NaCl, potassium acetate, etc) and alcohol type (isopropanol, methanol, ethanol, etc.)? Also, what role does pH of the solution play?



Replies:
For routine precipitation of nucleic acids, you need enough salt to neutralize the repulsion among the negatively charged strands of DNA, and enough alcohol to pull water molecules out of the DNA strand. In practice, this can be accomplished by doing any of the following: Measure the volume of the DNA solution and add...

1. One tenth volume of a 3.0 Molar NaAcetate, pH 7-8 stock, (0.3 Molar final) and 2.5 volumes of 95% ethanol.

2. One tenth volume of a 3.0 Molar NaAcetate pH 7-8 stock, (0.3 Molar final) and 2 volumes of absolute (100%) ethanol.

3. One tenth volume of a 3.0 Molar NaAcetate pH 7-8 stock, (0.3 Molar final) and one volume of isopropanol.

Variations: Ammonium acetate also works fine at the above concentration, as does NaCl. NaAcetate is probably used most commonly in routine lab work. As I recall, LiCl and some of the other salts have been used primarily where workers were trying to selectively precipitate different types of nucleic acid preferentially, e.g., small tRNAs. The pH is not critical, but should be in the neutral range for DNA. Sometimes RNA is precipitated with a slightly lower pH of 6.0, but this is not critically important for routine work.

If a lot of nucleic acid is present, you should see the precipitate form immediately. For very small amounts, centrifuging at 10,000 x g for 20 minutes will do the trick. Use either plastic microfuge tubes or high quality Corex tubes that won't break under the stress.

Paul Mahoney, Ph.D.



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