【正文】 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。 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。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), 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。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。 b) extractions with anic solvents。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 diversity of ecologically important microbes by repPCR genomic fingerprinting 39. Genomic Fingerprinting of Microanisms by AFLP(TM) and ERICanchor PCR 40. The use of pulsedfield gel electrophoresis to study bacteria recovered from the environment 41. Easy individual strain and munity typing by rDNA ITS1 analysis 42. In situ PCR methodologies for visualization of microscale geic and taxonomic diversities of prokaryotic munities 43. Sensitive multicolor fluorescence in situ hybridization for the identification of environmental microanisms 44. Use of Cloned Artificial Targets for FISH (catFISH) for the optimization of oligonucleotide probe hybridization conditions with 16S rRNA clones for in situ quantification of uncultivated prokaryotic cells 45. Denaturing gradient gel electrophoresis (DGGE) in microbial ecology 46. Fungal Community Analysis using PCR Denaturing Gradient Gel Electrophoresis (DGGE) 47. The Analysis of Microbial Communities with Terminal Restriction Fragment Length Polymorphism (TRFLP) 48. Microbial munity analysis by PCRsinglestrand conformation polymorphism (PCRSSCP) 49. Isolation of high molecular weight genomic DNA from soil bacteria for genomic library construction 50. Use of Biolog(R) for the Community Level Physiological Profiling (CLPP) of environmental samples 51. Fluorescent staining of microbes for total direct counts 52. Detection of microbes by Scanning Confocal Laser Microscopy (SCLM) 53. Production of antimicrobial antibodies and their utilization in studi