【正文】 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 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) technique. This method is often used in procedures for extracting nucleic acids directly from environmental samples, such as soil and sediment [22]. Such a treatment enhances bacterial cell disruption (., particularly species producing protective capsular slime and those involved in the formation of biofilms) by inducing phase changes in cell membranes through successive, rapid, extremes in temperature which render cells more susceptible to enzymatic and detergent lysis. Nucleic acid extractions The isolation of DNA from cells (., selectively eliminating other cellular ponents except the DNA) is the most straightforward of the three general steps. The methods of choice for extractions, traditionally, have involved the application of anic solvents (., phenol 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。mpl1 and Margit Mau1 (1) Division of Microbiology, GBF – German National Research Centre for Biotechnology, Mascheroder Weg 1, D38124, Braunschweig, Germany Introduction The development of methodologies for the analysis of microanisms and microbial ecology, at the molecular level (., nucleic acids, proteins, lipids, and their genes), has progressed phenomenally in recent years. Each methodology has specific advantages and disadvantages, or plications. However, the advances in PCR, cloning, gene probing, sequencing and fingerprinting have enabled techniques exploiting nucleic acids to be utilised extensively for the analysis of microanisms. Often, such protocols require, firstly, that the nucleic acids are extracted in a form which can be employed for the analyses. This may, in some cases, be more difficult than anticipated initially, since many bacteria are extremely resistant to cell disruption. Typically, these are Grampositive bacteria (., Mycobacterium spp., Peptococcus spp., Rhodococcus spp., etc.), as well as some Archae (., metha