谁能告诉我做活性污泥的种群分析DGGE前DNA提取的详细步骤？

MATERIALS AND METHODS
Soils. Eight soils were used to evaluate the efficiency of DNA extraction andpurification procedures. Six of these soils had been selected from a global soilcollection (9) to represent a range of soil properties (Table 1). The other two
soils， Native Kellogg (NK) and Cultivated Kellogg (CK)， were obtained from theNational Science Foundation Long-Term Ecological Research site at Kellogg Biological Station near Kalamazoo， Mich. NK and CK soils were from the samesoil series (Kalamazoo sandy loam， typic hapludalf)， but CK soil has been cultivatedfor the last 40 years while NK soil has been undisturbed. All soils came
from regions having predominantly luvisolic soils， as described under the Food and Agriculture Organization Soil Classification System (7). The six soils from the global collection had been sampled between 5 and 30 cm in depth in 1993.
NK and CK soils were sampled between 0 and 15 cm in 1993 and 1994. All soils were kept on ice or stored at 48C until they were tested in the laboratory.
Soil moisture contents were determined by drying at 1108C for 48 h. Particle size analyses were performed by a modified hydrometer method， in which the clay content was determined after 8 h (4). Carbon and nitrogen contents were
determined on oven-dried， ground samples in a Carlo Erba NA 1500 series 2 nitrogen/carbon analyzer (Fisons Instruments， Beverly， Mass.). Soil pH was determined in a slurry (5 parts distilled water， 1 part soil). Soil color was evaluated by visual examination in outdoor sunlight with Munsell color plates.
Bacterial strains and soil inoculation. Pseudomonas sp. strain B13 (5) was used as the seed organism. Cells were grown to late exponential phase on M9 medium supplemented with trace minerals and 5 mM 3-chlorobenzoate and
resuspended in 2 ml of extraction buffer (see below) before being inoculated into the soils. This cell suspension was mixed with sterilized soils， which were obtained by autoclaving twice at 1218C for 60 min. Seeded soils were kept at room temperature for 30 min prior to DNA extraction.
Effect of CTAB and PVPP on humic contamination of crude extracts. Hexadecylmethylammonium
bromide (CTAB) and polyvinylpolypyrrolidone (PVPP)
have been used in previous studies to complex and remove contaminants fromDNA (1， 10). Seeded NK soils were used to evaluate the effect of CTAB and
PVPP in the extraction buffer on humic contamination in crude extracts. Soil (5
g) was mixed with extraction buffer (see below) containing (i) no CTAB， no PVPP; (ii) 1% CTAB， no PVPP; or (iii) no CTAB， 2 g of PVPP. Soil suspensions were then processed by the extraction method described below. Spectrophotometric
A260/A280 and A260/A230 ratios were determined to evaluate levels of protein and humic acid impurities， respectively， in the crude extracts (14， 20).
SDS-based DNA extraction method. Since CTAB performed better in reducing humic contamination， it was used in the buffer for sodium dodecyl sulfate (SDS)-based DNA extraction. Soil samples of 5 g were mixed with 13.5 ml of DNA extraction buffer (100 mM Tris-HCl [pH 8.0]， 100 mM sodium EDTA [pH 8.0]， 100 mM sodium phosphate [pH 8.0]， 1.5 M NaCl， 1% CTAB) and 100 ml of proteinase K (10 mg/ml) in Oakridge tubes by horizontal shaking at 225 rpm for 30 min at 378C. After the shaking treatment， 1.5 ml of 20% SDS was added， and the samples were incubated in a 658C water bath for 2 h with gentle end-over-end inversions every 15 to 20 min. The supernatants were collected after centrifugation at 6，000 3 g for 10 min at room temperature and transferred into 50-ml centrifuge tubes. The soil pellets were extracted two more times by adding 4.5 ml
of the extraction buffer and 0.5 ml of 20% SDS， vortexing for 10 s， incubating at 658C for 10 min， and centrifuging as before. Supernatants from the three cycles of extractions were combined and mixed with an equal volume of chloroformisoamyl alcohol (24:1， vol/vol). The aqueous phase was recovered by centrifugation
and precipitated with 0.6 volume of isopropanol at room temperature for 1 h. The pellet of crude nucleic acids was obtained by centrifugation at 16，000 3 g for 20 min at room temperature， washed with cold 70% ethanol， and resuspended
in sterile deionized water， to give a final volume of 500 ml.
Cell lysis and direct microscopic counts. Cell lysis efficiency was estimated for six of the eight soils by direct microscopic counts of soil smears (3) obtained before and after the DNA extraction treatment. Before DNA extraction， 5 to 15 g of soil was blended in a Waring blender for 1 min in 150 to 190 ml of sterile，
filtered (0.2 mm)， deionized water. The coarse particles in the blended slurry were allowed to settle for 1 min， and then a 10-ml subsample was removed from the upper portion of the slurry and transferred to a 15-ml sterile tube. The
subsample was vortexed for 10 s， and 4- or 6-ml aliquots were removed for smears. Each aliquot was spread evenly in a 7-mm circle on a coated slide(Cel-Line Associates， Newfield， N.J.) and quickly dried at 408C. After DNA
extraction， smears were prepared from the remaining soil pellet and the pooled
supernatants from each sample. A subsample of soil pellet was removed to determine the moisture content， and the remaining pellet was blended with
appropriate amounts of water to prepare smears. Before DNA was precipitated and quantified from each supernatant， a 100-ml subsample of the supernatant was diluted 1:20 in water to make smears.
Dried smears were flooded with 10 ml of a fluorescent staining solution containing
2 mg of DTAF [5-(4，6-dichlorotriazin-2-yl)amino fluorescein; Sigma Chemical Co.， St. Louis， Mo.)] per 10 ml of buffer (0.85% NaCl， 50 mM
Na2HPO4 [pH 9.0]) (16). Flooded slides were held for 30 min in a covered container to prevent drying. The slides were rinsed by immersion in fresh buffer three times for 20 min each， rinsed with water after the final immersion， and air
dried. Slides were stored in the dark at 48C for no longer than 48 h before microscopic analysis.
The slides were examined with a 633 objective on a Leitz Orthoplan 2 epifluorescence microscope， a Lep HBO 50 mercury lamp， and a Leitz I3 filter
block (BP 450-490 excitation filter， RKP 510 beam splitter， and LP 515 suppression filter). A charge-coupled device camera (Princeton Instruments， Trenton，N.J.) was used to obtain digitized images of smears (26) by photographing fields selected at random along two central transects (8). Images were transferred to a
Power Macintosh 7100/66 via an ST135 etector/controller and GPIB interface
card (National Instruments， Austin， Tex.) for display by IP Lab Spectrum image analysis software (Signal Analytics Corp.， Vienna， Va.). Bacterial cells were counted by visual examination of images on the computer monitor. All counts were obtained by one investigator.
DNA extraction from gram-positive bacteria. The gram-positive bacteria used for comparing cell lysis methods were grown on tryptic soy agar at 378C overnight.
Three methods were used to evaluate DNA extraction from these bacteria in terms of DNA yield and fragment size: (i) grinding， freezing-thawing， and SDS; (ii) freezing-thawing and SDS; and (iii) SDS. Cell pellets from pure cultures
(15 ml; 1010 cells ml21) were ground with a mortar and pestle in the presence of sterile sand and liquid nitrogen before addition of extraction buffer and SDS. The preparations were then frozen at 2708C and thawed by microwave heating until they boiled briefly， a total of three times; then the DNA was extracted by following a protocol similar to the one described above. The DNA
yield was determined by spectrophotometry.
Purification of crude DNA extracts. Four methods for purifying small portions of crude extracts were evaluated. One-tenth to one-fifth of the crude DNA extract from 5 g of soil was processed in four ways， and the final volumes of the
eluates were adjusted to their original volumes. The four methods were as follows: (i) single minicolumn (extract was passed through one Wizard minicolumn containing 1 ml of Wizard PCR Preps purification resin [Promega， Madison，
Wis.]); (ii) double minicolumn (eluate from the first minicolumn was purified further with fresh resin and passage through another minicolumn); (iii) gel plus minicolumn (extract was purified by agarose gel electrophoresis followed by
passage of the excised and melted gel band through a Wizard minicolumn); and(iv) gel plus centrifugal concentrator. (The crude extract was subjected to gel electrophoresis， and the DNA band was excised， melted， and treated with Gelase
[Epicentre Technologies， Madison， Wis.] by following the rapid protocol of the manufacturer. Nucleic acids were then washed and concentrated in a Centricon-50 [Amicon Corp.， Beverly， Mass.].)
Since the minicolumn capacity was limited to 1 ml of resin， only a fraction (1/5 to 1/10) of the crude extract from 5 g of soil could be purified at a time. A larger-scale gel-plus-column purification procedure was used to purify the entire crude extracts from 5-g samples of the eight test soils. The procedure employed
a 20-ml-capacity column with a different resin (Wizard Minipreps Plasmid Purification
resin [Promega]) because the resin used in the minicolumns contained a 230-nm-absorbing substance that interfered with spectrophotometric measurement of DNA. The DNA was eluted from the resin twice with 500 ml of hot (708C) Tris-EDTA buffer to facilitate release of high-molecular-weight DNA.
DNA quantification. After small-scale rifications， DNA was quantified by
fluorometry with a TK 100 fluorometer (Hoefer Scientific Instruments， San Francisco， Calif.)， by following the extended assay protocol provided by the manufacturer. The fluorometer was calibrated with herring sperm DNA (Boehringer
Mannheim， Indianapolis， Ind.). The DNA yields were estimated on the basis of at least three replicate determinations.
DNA was quantified after large-scale purification by determining fluorescence intensities of extracts in agarose gel bands in scanned Polaroid photographs.
Crude and final DNA extracts were subjected to electrophoresis in Tris-acetate-EDTA (TAE) buffer containing 0.5 mg of ethidium bromide per ml in 0.7% agarose gels containing DNA standards of 5 to 60 ng of lambda phage DNA (Boehringer Mannheim). Gel photographs were scanned with a Hewlett-Packard ScanJet IIc scanner， generating digitized images that were analyzed with IP Lab
Spectrum software. A standard curve of DNA concentration (10 to 50 ng of DNA) versus integrated pixel intensity (r2 5 0.98) was prepared for each gel and used to calculate the final DNA concentrations in the DNA extracts.
PCR， restriction enzyme digestion， and Southern blotting. Primers and PCR conditions for amplifying 16S rRNA and clcD genes were described previously (28， 29). Restriction enzyme digests were performed with approximately 0.2 mg
ofDNA and 4 U of an endonuclease (BamHI， DraI， EcoRI， EcoRV， HindIII， or XhoI) in 20 ml of the appropriate buffer as provided by the manufacturer. After incubation for 12 to 16 h， the DNA fragments were resolved in a 1% agarose gel.
To evaluate DNA hybridization， 0.5 mg of the purified DNA from the seeded soils was digested with 10 U of BamHI， separated in an 0.8% agarose gel， and transferred to a GeneScreen Plus membrane (Dupont， Boston， Mass.). Prehybridization， hybridization， and washings were carried out as described previously
(29). A PCR-amplified 615-bp fragment of the clcD gene was used as the probe.