【正文】 nogens), with thick cell walls of polysaccharide or pseudopeptidoglycan, and many species of fungi and algae. General considerations Several protocols have been developed and described for the preparation of genomic DNA from bacteria, beginning with the prototypal method of Marrnur [16], which involved: a) cell disruption by an enzymedetergent lysis。C. The efficiencies of recovery, by centrifugation (12,000 g, 6176。C, 10 minutes. – Extract suspension with an equal volume (approximately 800 μl) chloroform/isoamyl alcohol (24:1) solution. Centrifuge (10,000 179。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。C, 2 minutes. Add 80 μl CTAB/NaCI solution (preheated at 65176。 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。Molecular Microbial Ecology Manual 1. Simplified protocols for the preparation of genomic DNA from bacterial cultures 2. Extraction of ribosomal RNA from microbial cultures 3. Extraction of microbial DNA from aquatic sources: Marine environments 4. Extraction of microbial DNA from aquatic sources: Freshwater 5. Methods for extracting DNA from microbial mats and cultivated microanisms: high molecular weight DNA from French press lysis 6. Extraction of microbial DNA from aquatic sediments 7. Extraction of microbial RNA from aquatic sources: Marine environments 8. Extraction of total RNA and DNA from bacterioplankton 9. Methods for extracting RNA or ribosomes from microbial mats and cultivated microanisms 10. Cell extraction method 11. DNA and RNA extraction from soil 12. Rapid simultaneous extraction of DNA and RNA from bulk and rhizosphere soil 13. Direct Extraction of Fungal DNA from Soil 14. Purification of microbial genes from soil and rhizosphere by magic capture hybridization and subsequent amplification of target genes by PCR 15. Direct ribosome isolation from soil 16. DNA Extraction from Actinorhizal Nodules 17. Quantification of nucleic acids 18. Quantification of nucleic acids from aquatic environments by using greenfluorescent dyes and microtiter plates 19. Degradation and turnover of extracellular DNA in marine sediments 20. Incorporation of thymidine into DNA of soil bacteria 21. Preparation of radioactive probes 22. Detection of Nucleic Acids by Chemiluminescence 23. Parameters of nucleic acid hybridization experiments 24. Detection and quantification of microbial DNA sequences in soil by Southern and dot/slot blot hybridization 25. Detection of microbial DNA sequences by colony hybridization 26. Polymerase chain reaction analysis of soil microbial DNA 27. Detection of microbial nucleic acids by polymerase chain reaction in aquatic samples 28. Isolation and detection of bacterial DNA sequences in dairy products 29. Quantitative PCR of environmental samples 30. Molecular beacons for homogeneous realtime monitoring of amplification products 31. Detection and enumeration of soil bacteria using the MPNPCR technique 32. Detection of mRNA and rRNA via reverse transcription and PCR in soil 33. Amplification of ribosomal RNA sequences 34. Cloning 16S rRNA genes and utilization to type bacterial munities 35. SARST, Serial Analysis of Ribosomal Sequence Tags 36. Oligonucleotide Fingerprinting of Ribosomal RNA Genes (OFRG) 37. Genotyping of bacterial isolates from the environment using LowMolecularWeight RNA fingerprints 38. Characterization of the di