【正文】 thyl ammonium bromide (CTAB), a cationic detergent, has been used extensively in the preparation of nucleic acids from fungi and plants, when large amounts of polysaccharide materials tend to interfere with the extraction. However, CTAB also has been proven useful for DNA extractions from bacterial cells by denaturing and precipitating the cell wall lipopolysaccharides and proteins [12]. In the presence of monovalent cation (., Na+) concentrations above M, DNA will remain soluble. Nonpolar detergents, including the Triton X series, Tween series, Nonidet P40, etc., are generally “milder” solubilising agents than the polar detergents and they seem to have a much more limited ability to initiate the disruption of bacterial cells. Cell disruption by “physical” methods Bacteria whose cell walls are not susceptible to enzymatic and detergent treatments may be disrupted using “harsher” (., also on the DNA) methods which may be described, arbitrarily, as “physical” or “mechanical” [ 10,11,14,19]. Such methods generate DNA which is often sheared and usually not of the relatively uniform, large, molecular weight that can be attained using enzymatic and detergent disruption. Thus, such methods may not be appropriate for preparing DNA for specific analytical techniques. However, in instances wherein it has not been critical that the DNA be of uniform high molecular weight, methods employing a French pressure cell or a sonicator have been used with success. The use of glass particles with the (mini)bead beater is particularly effective for disrupting most bacteria and is the method of choice for the preparation of DNA from bacterial cells in problematic matrices (., soils) [23]. Additionally, a method for the production of high molecular weight DNA from Grampositive and acidfast bacteria using a microwave oven has been described [1]. However, the efficacies of such methods, all of which require additional, specialised, equipment, have been limited, in most cases, in the range of bacteria for which a given method can be applied. A further application which has been shown to be effective, particularly in bination with other steps, for disrupting extremely recalcitrant bacteria is the freeze (in liquid nitrogen) and fast thaw (at 95– 98176。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, 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 37176。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。 g), the recovery of DNA from dilute suspensions may require centrifugations for as long as 30 minutes (Fig. 2). Figure 1 The recovery of DNA as a function of the precipitation temperature. Precipitations of varying amounts ( ng–010 μg) of DNA at extremly low temperatures (., ?70176。C to allow the DNA to be resuspended pletely. – Estimate the concentration of DNA in suspension by spectrophotometric measurement at 260 nm. For doublestranded DNA suspensions, at a wavelength of 260 nm and using a cuvette with a 1 cm light path, an OD of is equal to a concentration of 50 Mg/ml. The quality of the DNA can be estimated by measure ments of the A260/A280 and the A260/A230 ratios. The size of the DNA can be estimated by agarose gel (%, w/v) electrophoresis, subsequent staining with ethidium bromide and visualisation by . illumination. DNA of uniform size (approximately 20 kb) indicates that the DNA has been extracted without excessive shearing. DNA which has been sheared or degraded by nucleases will appear as a broad smear, of smaller molecular weight products. – Adjust the DNA suspension to a final stock concentration (., 1– 10 μg/μl before using an aliquot f or a PCR. – After adding TE buffer, some cells may begin to lyse and vortexing will induce shearing of released DNA. However, in the case of most bacteria, vortexing at this point will not produce noticeable shearing. – Many bacteria will lyse without using lysozyme. However, in many cases, lysozyme will facilitate lysis and, if it is used, it should be added before the Proteinase K and SDS. Many bacterial species will lyse quickly, but others may require longer incubation times. In some cases, overnight incubations, supplemented with additional Proteinase K and SDS, have proven successful in lysing the cells when shorter incubation times were not effective. K+ should be excluded from all buffers when SDS is used, as the detergent will precipitate, except at elevated temperatures. – It is important that the NaCI solution be well mixed with the lysate before adding the CTAB/NaCI solution, as the nucleic acids will precipitate (at room temperature) with the CTAB if the total Na+ concentration is below approximately M. – A ml micropipetter can be used, but the end of the pipette tip should be cut off to help prevent excessive shearing when pipet