【正文】 enol and chloroform) [13], which interact with hydrophobic ponents of protein and lipoprotein, causing denaturation. It is believed that forces maintaining the hydrophobic interiors of proteins, through their native conformations, are overe by exposure to hydrophobic solvents, resulting in the unfolding and precipitation of the protein [6]. The precipitate of denatured cellular material remains within the anic phase, which is separated by centrifugation. In general, phenol is an effective denaturing agent of protein, while chloroform will be more effective for polysaccharide materials. Thus, for the extraction of DNA from bacterial cells, mixtures of phenol/chloroform are more effective than either is, alone. Phenol of high purity (., redistilled), saturated and equilibrated with buffer (pH 8) should be used for the extractions. Recovery of DNA The standard method for recovering DNA from cell lysates and suspensions is by the use of alcohol (., ethanol or isopropanol) reversible denaturation (., the helical structure is extensively destroyed) and subsequent precipitation [5], followed by centrifugation. It is recognised that DNA precipitates poorly in saltfree solutions and that alcohol precipitations should be performed in the presence of a monovalent cation with a concentration of, at least, M. Precipitation of DNA in suspensions is initiated by adding suspension volume of 3 M sodium acetate (pH ) and – suspension volumes (calculated after the addition of salt) of 100% ethanol (for DNA suspensions of low concentration, a higher ratio of ethanol to suspension volume will facilitate DNA precipitation) [21]. Alternatively, volumes of M ammonium acetate (pH 8) can be used instead of sodium acetate [3]. In this case, small nucleic acid fragments (approximately 150 nucleotides and smaller), will not be precipitated, which may be advantageous in some cases. Isopropanol (– volumes) may be used, rather than ethanol, particularly when small volumes (., less than ml) are needed. Although it has bee an accepted practice to carry out DNA precipitations at extreme cold temperatures (., ?70176。C) and, in fact, may be counterproductive [27] (Fig. 1). Further, while the majority of DNA in concentrated suspensions is recovered quickly (., within 5 minutes) by centrifugation (12,000– 15,000 179。C) are less efficient than at 0176。C), were also observed to be dependent upon the amounts of DNA in suspension. The values indicated in the graph represent the means, calculated from the observed recoveries from suspension, of varying amounts of DNA. The ranges of observed recoveries are indicated, with the lowest and highest recoveries, at each temperature tested, and correspond to the lowest and highest concentrations of DNA, respectively. The graph was prepared from data taken from Zeugin and Hartley, 1985 [27]. Figure 2 The recovery of DNA as a function of the centrifugation time. The recovery of varying amounts ( ng–10 μg) of DNA is enhanced by increased centrifugation times. The efficiencies of recovery, by centrifugation (12,000 g, 6176。 g for 10 – 15 minutes. In general, latelog growth phase cultures should not be used for preparing DMA, as nucleases tend to accumulate in older cultures. Alternatively, bacterial colonies grown on agar media may be washed off the agar and collected in an Eppendorf tube. The Bacteria Washing Buffer should not contain EDTA, as some bacteria (., some Gramnegative species) will begin lysing upon exposure to chelating agents. After pelleting the cells, the medium is poured off and the rim of the tube is blotted with a paper towel to get rid of residual liquid. The bacterial pellet should weigh approximately g (wet weight), which should provide 40– 200 mg of DNA, depending upon the species of bacteria and the growth conditions. If there is more than g per tube, the cell pellet should be resuspended with Bacteria Washing Buffer and redistributed accordingly into additional Eppendorf tubes. This is not unimportant, since the efficiency of the extraction decreases with increasing cell material. With experience, one can estimate reliably the mass of the cell pellet from its size. Steps in the protocol – Resuspend the cell pellet (approximately g) pletely with 564 μl TE buffer (use a sterile toothpick to mix the pellet and ensure plete resuspension). – Add approximately 10 μg lyso zyme (crystalline) to the cell suspension (from this point, do not vortex!). Mix thoroughly by inverting the Eppendorf tube several times. Incubate 10– 60 minutes at 37176。C until the suspension bees relatively clear and viscous. – Add 100 μl NaCI (5 M) and mix thoroughly (do not vortex!). Incubate suspension at 65176。C, use a pipette tip wit h the tip cut off to pipette the viscous CTAB/NaCI solution) and mix thoroughly (do not vortex!). Incubate suspension at 65176。 g, 5 minutes) Transfer the upper (aqueous) phase (Supernatant 1), containing the nucleic acids, into a separate ml Eppendorf tube. – Extract Supernatant 1 with an equal volume (approximately 800 μl) of phenol/chloroform/isoamyl alcoh ol (25:24:1) solution. Centrifuge (15,000 179。 g, 5 minutes). Transfer the upper (aqueous) phase (Supernatant 3), containing the nucleic acids, into a separate ml Eppendorf tube. – Add volumes (approximately 560) isopropanol to precipitate nucleic acids. Mix gently by inverting the tube several times – the DNA should appear as a white, viscous, precipitate. Let sit at room temperature for 5 minutes to 1 hour. Centrifuge (12,000– 15,000 179。 g, 15 – 30 minutes at room temperature. Carefully remove the EtOH and blot the rim of the tube with a paper towel to get rid of excess liquid. – Briefly (not more than 5 minutes) dry pellet in a speedvac. – Resuspend each pellet in 50– 60 μl TE Buffer. Let sit at 3