【正文】 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 pipetting the aqueous phase containing the DNA. – Older, oxidised, phenol solutions should not be used as they may cause “nicking” of the DNA. The phenol solution should contain an antioxidis。 g 15 – 30 minutes) at room temperature. The DNA should be visible as a pellet on the side of the Eppendorf tube. Remove the isopropanol carefully, so as to avoid disturbing the pellet. – Wash the pellet with 500 μl EtOH (70%) by inverting the tube several times. Centrifuge 12,000– 15,000 179。 g, 5 minutes). Transfer the upper (aqueous) phase (Supernatant 2), containing the nucleic acids, into a separate ml Eppendorf tube. – Extract Supernatant 2 with an equal volume (approximately 800 μl) chlor oform:isoamyl alcohol (24:1) solution. Centrifuge (10,000 179。C, 10 minutes. – Extract suspension with an equal volume (approximately 800 μl) chloroform/isoamyl alcohol (24:1) solution. Centrifuge (10,000 179。C, 2 minutes. Add 80 μl CTAB/NaCI solution (preheated at 65176。C. Add 6 μl Proteinase K (10 mg/ml), and 30 μl SDS (10– 20%). Mix thoroughly (do not vortex!). Incubate at 37176。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 centrifugation time tested, and correspond lowest and highest concentrations of DNA, respectively. The graph was prepared from data taken from Zeugin and Hartley, 1985 [27]. An important consideration to keep in mind throughout the extraction process is the relationship between the amount of DNA in suspension and the ability, ultimately, to recover it. In studies to determine the optimal conditions for the recovery of DNA from suspensions by precipitation and centrifugation [27], the amount of DNA recovered was observed to be proportional to the concentration in suspension (Fig. 3). Thus, it is important to consider this relationship when deciding upon the extraction protocol to use and subsequent handling of the DNA. Figure 3 The recovery of DNA as a function of the amount of DNA in suspension. The recovery of DNA was observed to be dependent on the concentrations of the suspensions. The values indicated represent the means, calculated from the observed recoveries from suspension, after varying centrifugation times. The ranges of observed recoveries are indicated, with the lowest and highest recoveries, for each DNA concentration, corresponding to the shortest and longest centrifugation times (5–30 minutes). The graph was prepared from data taken from Zeugin and Hartley, 1985 [27]. Procedures The specific methods described here are simplified, rapid, protocols observed to be effective for isolating genomic DNA, from a wide range of bacteria, of a quality applicable for PCR. Protocol I – CTAB protocol for the extraction of bacterial genomic DNA This protocol is derived from the “miniprep” method described by Wilson [25]. Broth cultures (2– 5 ml) grown to midlog growth phase are harvested in ml Eppendorf tubes by centrifugation in a microfuge at 10,000– 15,000 179。C. The efficiencies of recovery, by centrifugation (12,000 g, 6176。 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), data suggest that precipitations at such temperatures present no significant advantage over precipitations carried out in ice water (., approximately 0176。 and c) recovery of the DNA by