CN1952150A - Method for extracting high molecular genome from bacterium - Google Patents

Abstract

The invention discloses a method of extracting high molecular genome from the bacteria. The method contains the steps of: breaking cell wall using lysozyme +protease +SDS or protease +SDS, extracting in TE buffer (removing proteins and polysaccharides), and preciping, washing, drying and dissolving. The invention begins with the factors influencing extracted DNA fragment length and obtains high molecular DNA fragment longer than fragments extracted by conventional method. The genomic DNA extracted in the method has more than 40 kb with OD260/OD280 between 1.8 and 2.0 detecting by absorbance. The method has the advantages of good repeatability and simple operation and can meet the need of the above molecular operation.

Description

A kind of genomic method of high molecular of from bacterium, extracting

Technical field

The present invention relates to a kind of bacterial genomes extracting method, reach by this method and extract polymer, big fragment gene group, belong to the microbial molecular field of biology to satisfy the needs of many molecular biological analysis.

Background technology

Because bacterial growth speed is fast, simple in structure, be easy to cultivate, of a great variety, advantage such as it is wide to distribute, therefore, illustrating of many important vital movements all is that research object is carried out with the bacterium, and many production practice all realize as carrier with bacterium, bacterium is very important and Microbial resources widely as a class, at medicine, chemical industry, metallurgy, environmental protection, aspects such as resource have shown function and the value that can not be substituted.At present, the research to bacterium more and more has been deep into molecular level.As molecular cloning, rflp analysis, library construction, the development of these methods and technology is all had higher requirement to the dna fragmentation size.In general, make up genomic library, initial DNA length must be more than 40kb, otherwise enzyme is cut the both sides, back all with effective fragment of appropriate end seldom.And carry out RFLP and pcr analysis, DNA length can be as short as 25kb, more than the length, can guarantee to produce after enzyme is cut RFLP fragment (20kb is following) at this, and can guarantee to comprise the fragment (general 2kb is following) that PCR increases.

At present, the method for laboratory extraction bacterial genomes DNA mainly contains CTAB method, alkali extraction method.But these methods often can only adapt to Gram-positive or gram negative bacterium, lack versatility, and will make the genomic DNA fragment of extraction reach the above relatively difficulty of 40kb.

Summary of the invention

In order to solve the deficiency that exists in the existing bacterium extracting method, the invention provides a kind of genomic method of high molecular of from bacterium, extracting.

A kind of genomic method of high molecular of extracting from bacterium comprises broken wall, extracting (Deproteinization, polysaccharide etc.), precipitation, dry, the dissolving of washing, and specific embodiment is as follows:

Broken wall: crack cell walls and cytolemma and be the prerequisite that obtains genomic dna, because the otherness of the bacteria wall of Gram-positive and gram negative bacterium, thereby adopted different broken walls at two bacterioids

Method: gram-positive microorganism adopts N,O-Diacetylmuramidase+proteolytic enzyme+SDS, and gram negative bacterium is adopted proteolytic enzyme+SDS.

Gram-positive microorganism: with the TE damping fluid suspension bacterium of 400-500ul, add the N,O-Diacetylmuramidase of 45-50ul, 20mg/ml and 40-60ul, 20% SDS, 37 ℃ of temperature are bathed the Proteinase K that adds 6-7ul, 20mg/ml behind the 1h, and 37 ℃ of temperature are bathed 1h.

Gram negative bacterium: with the TE damping fluid suspension bacterium of 400-500ul, add 40-60ul, 20% SDS, 37 ℃ of temperature are bathed the Proteinase K that adds 6-7ul, 20mg/ml behind the 1h, and 37 ℃ of temperature are bathed 1h.

Extracting: the CTAB/NaCl solution that the bacterium behind the broken wall is added 170-220ul removes polysaccharide, adopts isopyknic chloroform/primary isoamyl alcohol, centrifugal removal albumen and polysaccharide and other mixture.

Precipitation: draw supernatant to centrifuge tube, to the Virahol that wherein adds 0.8-1.2 times of volume, deposit D NA.

Washing is dry: add the dehydrated alcohol of 0.8-1.2 times of volume, and mixing, abundant airing is avoided influence to subsequent operations to remove ethanol.

Dissolving: the aseptic deionized water or the TE dissolving DNA that add 30-50ul.

Described TE damping fluid is: 1mol/L TrisCl, and pH8.0,0.5mol/L EDTA, the 0.5mol/L citric acid adds water to 1000ml;

Described CTAB/NaCl solution is: 4.1g NaCl is dissolved in 80ml water, slowly adds 10g CTAB, adds water to 100ml;

Described chloroform is: primary isoamyl alcohol (24: 1), Virahol, 70% ethanol, 20%SDS, Proteinase K (20mg/ml), N,O-Diacetylmuramidase (20mg/ml).

The present invention starts with from the factor that the dna fragmentation size is extracted in influence, be respectively Gram-positive and gram negative bacterium, extracted conventional the big polymer dna fragmentation of the fragment of carrying, the genomic dna that extracts reaches more than the 40kb, through absorbance detection, OD260/OD280 ratio between 1.8-2.0, the method good reproducibility, simple to operate, can satisfy above-mentioned molecule manipulation fully.

The present invention adds EDTA and citric acid double sequestration divalent-metal ion and then suppresses to depend on the activity of the DNA enzyme of divalent-metal ion most possibly in the buffering system of Tris-HCl, DNA is played protection, prevents and suppresses the degraded of DNase to DNA; The present invention is cut into big mouth with the rifle head and avoids when DNA is in dissolved state, weakening the vortex of solution with the piping and druming back and forth of rifle head as far as possible, and the mechanical shearing that as far as possible reduces DNA in the solution destroys.

Description of drawings

Thiobacillus ferrooxidant (Gram-negative) genomic dna of Fig. 1: A. for extracting with conventional CTAB method; B. the thiobacillus ferrooxidant genomic dna of carrying for present method, Marker is λ DNA cuttedby HindIII;

Fig. 2: A. is Bacillus subtilus (Gram-positive) genomic dna that extracts with present method; B. be the bacillus subtilis gene group DNA with conventional CTAB method extraction, Marker is λ DNA cutted by HindIII.

Embodiment

Embodiment 1:

Collect the new fresh thalli of the about 1g of weight in wet base, suspend the centrifugal 2min of 10000rpm with TE;

Remove supernatant, and in precipitation, add 480ulTE, the suspension bacterium;

Add the 0.05ml N,O-Diacetylmuramidase in suspension, put upside down mixing, 37 ℃ of temperature are bathed 1h.(be fit to gram-positive microorganism, Gram-negative bacteria directly enters step 4);

Add 20%SDS and the 5ul Proteinase K of 40ul, put upside down mixing, 37 ℃ of temperature are bathed 1h;

Add CTAB/NaCl solution 200ul, the soft mixing of putting upside down, 65 ℃ of temperature are bathed 10min;

Add isopyknic chloroform: the soft mixing of putting upside down of primary isoamyl alcohol (24: 1), the centrifugal 5min of 12000rpm;

Supernatant liquor is transferred in the new centrifuge tube with careful absorption of the rifle head that is cut into big mouthful;

Add the soft mixing of putting upside down of isopyknic Virahol, choose floss with the sterilization toothpick and transfer to a new centrifuge tube;

With 70% washing with alcohol twice, airing;

The toothpick that is attached with DNA is transferred in the new centrifuge tube, added the dissolving of spending the night of 50ul sterilized water.Lysate is the DNA that is carried.

Claims (2)

1. one kind is extracted the genomic method of high molecular from gram-positive microorganism, and it is characterized in that: comprise broken wall, extracting, precipitation, washing drying, dissolving, specific embodiment is as follows:

Broken wall: with the TE damping fluid suspension bacterium of 400-500ul, add the N,O-Diacetylmuramidase of 45-50ul, 20mg/ml and 40-60ul, 20% SDS, 37 ℃ of temperature are bathed the Proteinase K that adds 6-7ul, 20mg/ml behind the 1h, and 37 ℃ of temperature are bathed 1h;

Extracting: the CTAB/NaCl solution that the bacterium behind the broken wall is added 170-220ul removes polysaccharide, adopts isopyknic chloroform/primary isoamyl alcohol, centrifugal removal albumen and polysaccharide and other mixture;

Precipitation: draw supernatant to centrifuge tube, to the Virahol that wherein adds 0.8-1.2 times of volume, deposit D NA;

Washing is dry: add the dehydrated alcohol of 0.8-1.2 times of volume, and mixing, abundant airing;

Dissolving: the aseptic deionized water or the TE dissolving DNA that add 30-50ul;

Described TE damping fluid is: 1mol/L TrisCl, and pH8.0,0.5mol/L EDTA, 0.5 mol/L citric acid adds water to 1000ml;

Described CTAB/NaCl solution is: 4.1g NaCl is dissolved in 80ml water, slowly adds 10g CTAB, adds water to 100ml;

Described chloroform is: primary isoamyl alcohol (24: 1), Virahol, 70% ethanol, 20%SDS, Proteinase K (20mg/ml), N,O-Diacetylmuramidase (20mg/ml).

2. one kind is extracted the genomic method of high molecular from Gram-negative bacteria, and it is characterized in that: comprise broken wall, extracting, precipitation, washing drying, dissolving, specific embodiment is as follows:

Broken wall: with the TE damping fluid suspension bacterium of 400-500ul, add 40-60ul, 20% SDS, 37 ℃ of temperature are bathed the Proteinase K that adds 6-7ul, 20mg/ml behind the 1h, and 37 ℃ of temperature are bathed 1h;

Extracting: the CTAB/NaCl solution that the bacterium behind the broken wall is added 170-220ul removes polysaccharide, adopts isopyknic chloroform/primary isoamyl alcohol, centrifugal removal albumen and polysaccharide and other mixture;

Precipitation: draw supernatant to centrifuge tube, to the Virahol that wherein adds 0.8-1.2 times of volume, deposit D NA;

Washing is dry; The dehydrated alcohol that adds 0.8-1.2 times of volume, mixing, abundant airing;

Dissolving: the aseptic deionized water or the TE dissolving DNA that add 30-50ul;

Described TE damping fluid is: 1mol/L TrisCl, and pH8.0,0.5mol/L EDTA, 0.5 mol/L citric acid adds water to 1000ml;

Described CTAB/NaCl solution is: 4.1g NaCl is dissolved in 80ml water, slowly adds 10g CTAB, adds water to 100ml;

Described chloroform is: primary isoamyl alcohol (24: 1), Virahol, 70% ethanol, 20% SDS, Proteinase K (20mg/ml), N,O-Diacetylmuramidase (20mg/ml).

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