Phenol Extraction and Precipitation of DNA

Phenol and chloroform extractions remove other macromolecules, such as proteins and lipids. The phenol should be of good quality and buffered for 

DNA is precipitated from aqueous solutions by ethanol or isopropanol in the presence of salt. The amount of alcohol and salt depends on the type of salt that one wishes to use (Table 6.2). The type of salt used depends largely on downstream applications for which the DNA is to be used. For example, precipitation in the presence of ammonium acetate removes small molecules such as nucleotides, and the DNA can be used for many enzymatic reactions. On the other hand, the phosphorylating enzyme, T4 kinase, is inhibited by ammonium ions, and unless DNA is reprecipitated in the presence of salts other than ammonium acetate, the phosphorylation reaction may be inhibited. For most routine purposes, alcohol precipitation of DNA with sodium acetate is preferred over sodium chloride because of the higher solubility of the acetate salt in ethanol. Selection of isopropanol or ethanol is more of a convenience than a rule. Although isopropanol precipitation requires an equal volume of isopropanol for the precipitation of DNA, that with ethanol requires 2 volumes and hence can increase the total volume 

Salt Salt Stock Solution Final Concentration 1 

Recovery of DNA from dilute solutions ( 10 pg DNA/mL) can be enhanced by the addition of an appropriate carrier substance before ethanol precipitation. Molecular biology grade glycogen, which is available commercially, is added to the sample at a concentration of 20 to 40 pg/mL before the addition of ethanol. The dilute DNA solutions can also be concentrated by repeated extraction with. vec-butanol (mixing the solution by inverting several times) followed by centrifugation at 3000 x g for 5 minutes at room temperature, discarding the butanol phase each time. Each extraction will extract water out of the solution and hence concentrate the DNA. Finally, DNA can be ethanol precipitated. 

Although ethanol precipitation of DNA is recommended for dilute DNA solutions, DNA in an appropriate concentration (ca. 0.5 pg/mL) can be dialyzed against several changes of TE until the OD270 nm of the dialysate is less than 0.05. The advantages of dialysis method are that DNA need not be dried and dissolved, which takes 1 to 2 days, and that there is lesser shearing of DNA. 

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