Biotechnology Index Glossary
Concentrating Nucleic Acids
The need to concentrate DNA or RNA from dilute, aqueous solution arises quite frequently, for example, in almost all protocols for purification of nucleic acids from cells. The method most commonly used for this purpose is precipitation with ethanol or isopropanol.
Precipitation of DNA
A routine protocol for precipitating DNA entails the following simple steps:
- Adjust the concentration of monovalent cations in the sample by adding 1/10th volume of 3 M sodium acetate to the sample.
- Add 2 volumes of ethanol and mix gently. If there is sufficient DNA in the sample, you will see a white precipitate form very rapidly.
- Recover the DNA by centrifugation, and rinse the resulting pellet with 70% ethanol to remove residual salt.
- Evaporate off residual ethanol and resuspend the DNA in the desired buffer to the desired concentration.
The image to the right shows samples of genomic DNA before and after addition of sodium acetate and ethanol. The precipitate became visible within a few seconds of adding ethanol. When the DNA in contained in small volumes, the procedures is usually carried out in 1.5 ml microcentrifuge tubes.
Like many procedures in molecular biology, there are several effective variations-on-a-theme for preciptating DNA from solution, and with a few exceptions, the choice of which to use is largely a matter of personal preference. The significant variables for nucleic acid precipitation are:
- Type and concentration of monovalent cation: The frequently-used sources of cations are:
- Sodium acetate at a final concentration of 0.3 M (3 M stock solution).
- Sodium chloride at a final concentration of 0.2 M (5 M stock solution).
- Ammonium acetate at a final concentration of 2.5 M (7.5 M stock solution)
- Lithium chloride at a final concentration of 0.8 M (8 M stock solution).
In some situations, one salt is preferred over another. For example, ammonium acetate should not be used if the DNA is going to be phosphorylated with polynucleotide kinase. If the DNA contains the detergent SDS, sodium chloride is the choice because it allows SDS to remain soluble in 70% ethanol.
- Ethanol versus isopropanol: Isopropanol is an effective alternative to ethanol and has the advantage of precipitating DNA at lower concentrations. Instead of mixing 2 volumes of ethanol with the DNA-salt solution, addition of one volume of isopropanol will suffice.
- Time and temperature allowed for precipitation: Precipitation occurs very rapidly except when DNA content is very low (i.e. < 100 ng). In most cases, you can start centrifugation immediately after adding ethanol or isopropanol. Many older manuals indicate that precipitation should be allowed to occur in the cold (e.g. -20C). This has been shown to be unnecessary. Typically, centrifugation is at about 12,000 x g for 10 to 15 minutes.
- Drying DNA before resuspension: The purpose here is to allow evaporation of ethanol or isopropanol. It is best not to overdry the DNA, which makes re-solution more difficult and can cause denaturation of DNA. Drying is best achieved by leaving the open tube on the lab bench for a few minutes.
Precipitation of RNA
The methods used for precitation of RNA are essentially identical to those described above for DNA. The major concern in any type of RNA work is avoiding ribonuclease contamination. For ethanol precipitation of RNA, people generally use 2.5 to 3 volumes of ethanol, instead of the 2 volumes commonly used for precipitation of DNA.
Large RNAs, including mRNAs and ribosomal RNAs, become insoluble in solutions containing high concentrations of salt. This property can be exploited as an alternative method for precipitation and concentration of large RNAs. Most commonly, a solution containing RNA is mixed with an equal volume of RNase-free 8 M lithium chloride, and after incubation for > 2 hours on ice, the RNAs are collected by centrifugation. This method should not be used to prepare RNA for reverse transcription
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Last updated on January 21, 2000
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