CN102533730A - Method for separating and purifying large plasmids from bacteria - Google Patents

Abstract

The invention discloses a method for separating and purifying large plasmids from bacteria, and belongs to the technical fields of biochemistry and molecular biology. The method comprises the following steps of: embedding the bacteria in a low smelting point agarose gel; breaking the bacteria in the gel by using lysozyme, and digesting the protein of the bacterial in the gel by using proteinase; separating the DNA of the bacteria by using pulsed field gel electrophoresis; and recovering the gel to obtain the large plasmid DNA in the bacteria. The large plasmid DNA obtained through separation and purification has the characteristics of high purity, complete structure and the like. The method has important significance for researching biological characteristics and functions of the large plasmids and transforming and developing and utilizing the large plasmids in the bacteria.

Description

A kind of from bacterium the method for the big plasmid of separation and purification

Technical field

A kind of from bacterium the method for the big plasmid of separation and purification, belong to the Biochemistry and Molecular Biology technical field.Can be used for the extraction and the evaluation of the big plasmid of bacterium.

Background technology

Plasmid is the outer genetic elements that can carry out self-replacation of karyomit(e); Magnitude range is not from waiting more than the 1kb to 200kb; Extensively be present in the cell of all kinds of organisms, especially more common in bacterium, some nonessential biological character of coding bacteria live; Like sex fimbria, bacteriocin, toxin and resistance etc., general unconformability is to bacterial chromosome.Plasmid great majority commonly used now often contain antibiotics resistance gene through transformation or artificial constructed, are instruments important in the molecular biology recombinant DNA technology.Plasmid in the separation and Extraction bacterium is the research and the first step of transforming plasmid; Usually adopt alkaline lysis method of extracting plasmid DNA at 10kb with interior miniplasmids for size, its ultimate principle is: when thalline in NaOH and SDS solution during cracking, sex change takes place in protein and DNA; After adding neutralizer; Plasmid DNA molecule is stayed when centrifugal in the supernatant because less renaturation rapidly is dissolved state; Chromosomal DNA is owing to be not easy renaturation more greatly and be cotton-shaped with protein, precipitablely when centrifugal gets off.Therefore for the big plasmid in the bacterium, do not have miniplasmids so big, do not prove effective so traditional alkaline lysis is used for extracting the big plasmid of bacterium because its size is compared difference with karyomit(e).

Summary of the invention

In order to overcome the deficiency that traditional alkaline lysis can not be used to extract the big plasmid of bacterium, the invention provides a kind of simple and practical from bacterium the method for the big plasmid of separation and purification.This method utilizes the PFGE technology to combine the glue absorption method can effectively extract the large plasmid DNA in the bacterium.

The technical scheme that the present invention adopted is:

(1) culturing bacterium: the single colony inoculation of picking is in 4ml LB liquid nutrient medium, and centrifugal after 37 ℃ of vibrations (250rpm) incubated overnight (4000rpm, 10min, 4 ℃) are collected thalline.

(2) make blob of viscose: with PIV damping fluid (10mM Tris; 1M NaCl PH7.6) after the washing, gets the resuspended bacterium of 1ml PIV damping fluid; With the low melting-point agarose gel mixing (temperature remains on 50 ℃) of 1ml 2%, pour into blob of viscose and cast making bacterium blob of viscose in the groove.

(3) digestible protein: blob of viscose is put into fresh lysate Lysis buffer (6mM Tris, 0.1M EDTA, 1M NaCl; 0.5%Brij-58,0.2% Sodium desoxycholate, 0.5%SDS; RNaseA 20 μ g/ml; N,O-Diacetylmuramidase 1mg/ml) 37 ℃ of jogs spend the night in, and (0.5M EDTA 1%SDS) has removed protein with 50 ℃ of digested overnight of ESP damping fluid (ES that contains Proteinase K 100 μ g/ml) after the wash module to the ES damping fluid.

(4) electrophoretic separation: with TE damping fluid (10mM Tris-HCl, 1mM EDTA, PH8.0) the above-mentioned blob of viscose that spends the night through the digestion of ESP damping fluid of washing; The PFGE appearance carries out electrophoresis on the last blob of viscose, and deposition condition is: voltage 6V/cm, 120 ° of angles; Start transition time range: 1-10s, switching time, scope was 20-60s at last, the electrophoresis time scope is 6-24h; The electrophoresis liquid damping fluid is 0.5 * TBE, and temperature remains on 14 ℃.Electrophoresis after EB dyeing to observe big plasmid and chromosomal separation case.

(5) cutting glue reclaims:

1. cut the sepharose piece that contains the purpose large plasmid DNA with scalpel and weigh, add the heavy sol solutions QX1 of 3 times of glue earlier, add the heavy distilled water of 2 times of glue again.

2. vibrate and made QIAEX II particle resuspended in 30 seconds.If the DNA total amount in the blob of viscose is no more than 2 μ g, then in the sample sol solutions, add the QIAEX II particle of 10 μ l, 50 ℃ of insulation 10min then, vibration in per two minutes once makes particle resuspended, lets blob of viscose fully melt to discharge DNA be attached on the QIAEX II particle.This moment, the color of mixture should be yellow, if orange or purple, the sodium acetate solution that should add pH 5.0 3M is regulated its color to yellow, continues to be incubated simultaneously 5min at least again.

3. 13000rpm is centrifugal 30 seconds, draws supernatant carefully, abandons it.

4. add QX1 damping fluid 500 μ l, the resuspended particle that vibrates, washing granule, centrifugal 30 seconds of 13000rpm draws supernatant carefully, abandons it.This step is with the residual gel of flush away.

5. add 500 μ l damping fluid TE (10mM Tris-HCl, 1mM EDTA, PH8.0), the resuspended particle that vibrates, washing granule, centrifugal 30 seconds of 13000rpm draws supernatant carefully, abandons it.This step is with the residual salinity of flush away.

6. drying at room temperature 10-15 minute; Bleach up to particle; Add pH 8.5 10mM Tris-HCl damping fluids 20 μ l with the dissolving large plasmid DNA, get 2 μ l and carry out the gel electrophoresis of plain agar sugar to identify the purity of the DNA that is extracted, all the other are subsequent use in-20 ℃ of preservations.

Beneficial effect of the present invention: the present invention utilizes the PFGE technology to combine the large plasmid DNA in the effective separation and purification bacterium of glue absorption method ability, has overcome the drawback that traditional alkaline lysis can not be used to extract the big plasmid of bacterium.The present invention has remedied traditional alkaline lysis in the deficiency of extracting on the bacterial plasmid, has realized effective extraction of big plasmid in the bacterium.To the biological characteristics and the function of big plasmid in the research bacterium, and the big plasmid in transformation and the development and use bacterium, important role all had.

Description of drawings

Fig. 1. PFGE separates the electrophoretogram of the big plasmid pBSSB1 of Salmonella typhi GIFU10007;

Fig. 2. the electrophoresis evaluation figure of the big plasmid pBSSB1 that from Salmonella typhi GIFU10007, extracts.

Embodiment

Related linear plasmid pBSSB1 (Baker S, Hardy J, Sanderson KE among the following embodiment; Quail M; Goodhead I, Kingsley RA, Parkhill J; Stocker B, Dougan G (2007) A novel linear plasmid mediates flagellar variation in SalmonellaTyphi. Salmonella typhi GIFU10007 (Zhang H PLoS Pathog 3:e59.); Sheng X, Xu S et al (2009) Global transcriptional response of Salmonella enterica serovar Typhi to anti-z66 antiserum. FEMS Microbiol Lett 298:51-55) is stored in Jiangsu University's preclinical medicine and laboratory medicine institute of medical technology institute.

Embodiment: with separation and purification Salmonella typhi one linear plasmid pBSSB1 (27kb) is the example explanation:

Detailed process is:

(1) culturing bacterium: picking Salmonella typhi GIFU10007 ( S. single colony inoculation Typhi) is in 4ml LB liquid nutrient medium, and centrifugal after 37 ℃ of vibrations (250rpm) incubated overnight (4000rpm, 10min, 4 ℃) are collected thalline.

(2) make blob of viscose: with PIV damping fluid (10mM Tris; 1M NaCl PH7.6) after the washing, gets the resuspended bacterium of 1ml PIV damping fluid; With the low melting-point agarose gel mixing (temperature remains on 50 ℃) of 1ml 2%, pour into blob of viscose and cast making Salmonella typhi blob of viscose in the groove.

(3) digestible protein: the Salmonella typhi blob of viscose is put into fresh lysate Lysis buffer (6mM Tris, 0.1M EDTA, 1M NaCl; 0.5%Brij-58,0.2% Sodium desoxycholate, 0.5%SDS; RNaseA 20 μ g/ml; N,O-Diacetylmuramidase 1mg/ml) 37 ℃ of jogs spend the night in, and (0.5M EDTA 1%SDS) has removed protein with 50 ℃ of digested overnight of ESP damping fluid (ES that contains Proteinase K 100 μ g/ml) after the wash module to the ES damping fluid.

(4) electrophoretic separation: with the above-mentioned blob of viscose that spends the night through the digestion of ESP damping fluid of TE damping fluid washing, the PFGE appearance carries out electrophoresis on the last blob of viscose, and deposition condition is: voltage 6V/cm; 120 ° of angles; Start transition time 1s, last switching time 35s, electrophoresis time 18h; The electrophoresis liquid damping fluid is 0.5 * TBE, and temperature remains on 14 ℃.Electrophoresis is after EB dyes, and big plasmid and chromosomal separation case are as shown in Figure 1, successfully the linear big plasmid pBSSB1 of the 27kb in the Salmonella typhi has been realized separating with bacterial chromosome.

(5) cutting glue reclaims:

1. cut the sepharose piece of the big plasmid pBSSB1 among Fig. 1 with scalpel and weigh 200mg, add earlier the sol solutions QX1 of 600mg, add the distilled water of 400mg again.

2. vibrate and made QIAEX II particle resuspended in 30 seconds; The QIAEX II particle that in the sample sol solutions, adds 10 μ l immediately; 50 ℃ are incubated 10min then; Vibration in per two minutes once makes particle resuspended, lets blob of viscose fully melt to discharge DNA be attached on the QIAEX II particle, and this moment, the color of mixture be a yellow.

3. 13000rpm is centrifugal 30 seconds, draws supernatant carefully, abandons it.

4. add QX1 damping fluid 500 μ l, the resuspended particle that vibrates, washing granule, centrifugal 30 seconds of 13000rpm draws supernatant carefully, abandons it.This step is with the residual gel of flush away.

5. add 500 μ l damping fluid TE (10mM Tris-HCl, 1mM EDTA, PH8.0), the resuspended particle that vibrates, washing granule, centrifugal 30 seconds of 13000rpm draws supernatant carefully, abandons it.This step is with the residual salinity of flush away.

6. drying at room temperature is 10 minutes, and particle bleaches, and adds pH 8.5 10mM Tris-HCl damping fluids 20 μ l then and dissolves big plasmid pBSSB1, gets 2 μ l and carries out the gel electrophoresis of plain agar sugar, and all the other are subsequent use in-20 ℃ of preservations.As shown in Figure 2, present embodiment has successfully extracted the linear big plasmid pBSSB1 that highly purified size is 27 kb.

At last, it is pointed out that above what enumerate only is specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, the condition that can also pass through to change pulse electrophoresis is with big plasmid of all sizes in the separation and purification bacterium., all should think within protection scope of the present invention to the various effective distortion that above-mentioned embodiment carries out according to conception of the present invention.

Claims (1)

1. the method for the big plasmid of separation and purification from bacterium is characterized in that step is:

(1) culturing bacterium: the single colony inoculation of picking is in 4ml LB liquid nutrient medium, and is centrifugal after 37 ℃ of shaken overnight are cultivated, and collects thalline;

(2) make blob of viscose: with the PIV damping fluid (10mM Tris, 1M NaCl, PH7.6) wash after; Get the resuspended bacterium of 1ml PIV damping fluid; With the low melting-point agarose gel mixing of 1ml 2%, temperature remains on 50 ℃, pours into blob of viscose and casts making bacterium blob of viscose in the groove;

(3) digestible protein: blob of viscose is put into 37 ℃ of jogs of fresh lysate Lysis buffer spend the night, removed protein with 50 ℃ of digested overnight of ESP damping fluid after the ES damping fluid wash module; The composition of said Lysis buffer is 6mM Tris, 0.1M EDTA, 1M NaCl, 0.5%Brij-58,0.2% Sodium desoxycholate, 0.5%SDS, RNaseA 20 μ g/ml, N,O-Diacetylmuramidase 1mg/ml; Said ESP damping fluid is the ES damping fluid that contains Proteinase K 100 μ g/ml;

(4) electrophoretic separation: with the above-mentioned blob of viscose that spends the night through the digestion of ESP damping fluid of TE damping fluid thorough washing, the PFGE appearance carries out electrophoresis on the last blob of viscose, and deposition condition is: voltage 6V/cm; 120 ° of angles; Start transition time range: 1-10s, switching time, scope was 20-60s at last, the electrophoresis time scope is 6-24h; The electrophoresis liquid damping fluid is 0.5 * TBE, and temperature remains on 14 ℃; Electrophoresis after EB dyeing to observe big plasmid and chromosomal separation case;

(5) cutting glue reclaims:

1. cut the sepharose piece that contains the purpose large plasmid DNA with scalpel and weigh, add the heavy sol solutions QX1 of 3 times of glue earlier, add the heavy distilled water of 2 times of glue again;

2. vibrate and made QIAEX II particle resuspended in 30 seconds; If the DNA total amount in the blob of viscose is no more than 2 μ g; The QIAEX II particle that then in the sample sol solutions, adds 10 μ l; 50 ℃ of insulation 10min then, vibration in per two minutes once makes particle resuspended, lets blob of viscose fully melt to discharge DNA be attached on the QIAEX II particle; This moment, the color of mixture should be yellow, if orange or purple, the sodium acetate solution that should add pH 5.0 3M is regulated its color to yellow, continues to be incubated simultaneously 5min at least again;

3. 13000rpm is centrifugal 30 seconds, draws supernatant carefully, abandons it;

4. add QX1 damping fluid 500 μ l, the resuspended particle that vibrates, washing granule, centrifugal 30 seconds of 13000rpm draws supernatant carefully, abandons it, and this goes on foot with the residual gel of flush away;

5. add 500 μ l damping fluid TE, the resuspended particle that vibrates, washing granule, centrifugal 30 seconds of 13000rpm draws supernatant carefully, abandons it, and this goes on foot with the residual salinity of flush away;

6. drying at room temperature 10-15 minute; Bleach up to particle; Add pH 8.5 10mM Tris-HCl damping fluids 20 μ l with the dissolving large plasmid DNA, get 2 μ l and carry out the gel electrophoresis of plain agar sugar to identify the purity of the DNA that is extracted, all the other are subsequent use in-20 ℃ of preservations.

Images