CN102206627A - Reagent and method for extracting plasmid DNA - Google Patents

Abstract

The invention provides a reagent and a method for extracting plasmid DNA. The reagent comprises a thalline cracking reagent, a protein extracting reagent, a plasmid DNA precipitating reagent and a plasmid DNA dissolving reagent. With utilization of the method provided in the invention, the time for extracting plasmids is short and no more than 10 minutes is needed to extract a plasmid. The reagent provided in the invention has simple construction and uses common experimental reagents which are widely acceptable and cheap, and the experimental reagents at domestic standard of analytical purity can extract high yield and high purity plasmids, thereby substantially reducing experimental expense. The plasmid DNA obtained in the invention has high yield, good purity and no pollution to protein and RNA, and can be directly used for experiments of enzyme digestion, connection, order-checking, prokaryotic conversion, eukaryotic transfection, which enables the operation of biological experiments to be substantially convenient, time for experiment to be saved and experiment efficiency to be increased.

Description

A kind of reagent and method of extracting plasmid DNA

Technical field

The present invention relates to biology field, relate in particular to a kind of reagent and method of extracting plasmid DNA.

Background technology

Plasmid DNA is a genetically engineered carrier commonly used, can carry foreign gene enters and increases in bacterium, zooblast or the plant materials and express, the extraction of plasmid DNA and purifying are most widely used general and the most basic technology in the molecular biology research field, and efficient that plasmid extracts and quality have direct relation for the success or not of subsequent experimental (enzyme is cut, pcr amplification and order-checking etc.).At present at home and abroad, a lot of biological reagent companies have all developed and have extracted the relevant test kit of plasmid technology, as Pu Luomaige, day root biochemistry, living worker etc.Such test kit is many, and the cost height costs an arm and a leg based on purification column, and reagent is numerous and diverse and consuming time longer.If alkaline lysis, phenol-chloroform method for extracting with improvement extract plasmid, the plasmid DNA that obtains yields poorly, of low quality, can not satisfy the requirement that the enzyme in downstream is cut and checked order, plurality of reagents is mixed application simultaneously, strengthened the workload of experiment undoubtedly, making influences biological experiment operation and experimental result by the experimental period significant prolongation.

Summary of the invention

The invention provides a kind of reagent and method of extracting plasmid DNA, the reagent cost of the used extraction plasmid of the present invention is low, the method of extracting plasmid DNA is simple to operate, and the yield plasmid height that reagent of the present invention and method extract, purity height, quick, simple and easy, efficient, high-quality and advantage that cost is low that the present invention has.

For achieving the above object, the present invention adopts following technical proposals:

A kind of reagent that extracts plasmid DNA is characterized in that it comprises cellular lysate reagent, albumen extraction agent, plasmid DNA precipitation reagent and plasmid DNA solubilising reagent; The component of described cellular lysate reagent is: 10mM Tris-HCl, 1mM EDTA, 100mM sodium-chlor and 5mg/ml N,O-Diacetylmuramidase are solvent with the ultrapure water, and the pH value of described Tris-HCl is 7.4-8.0; The component of described albumen extraction agent is: volume ratio is 1: 1 phenol and a chloroform mixture; The component of described plasmid DNA precipitation reagent is: 5.5M sodium acetate soln and the dehydrated alcohol of PH7.5, and described sodium acetate soln and dehydrated alcohol volume ratio are 1: 3; The component of described plasmid DNA solubilising reagent is the ultrapure water that contains 0.1mg/ml RNase.

Wherein, the best pH value of described reagent Tris-HCl is 8.0.

The present invention also provides a kind of method of extracting plasmid DNA, and it may further comprise the steps:

(1) shakes bacterium amplification plasmid; The intestinal bacteria of carrying plasmid are inoculated in the LB solid medium, select the mono-clonal colony inoculation, be placed on the 30-38 ℃ of constant temperature shaking table with 150-250 rev/min of wave and culture in LB liquid nutrient medium amplification cultivation;

(2) get 12-16 hour OD of cultivation ₆₀₀Reach the good bacterium bacterium liquid 1.5ml of growth conditions more than 0.6 in the 1.5ml centrifuge tube, under 20 ℃-25 ℃ centrifugal 30 seconds, abandon supernatant with 13000g;

(3) add the described cellular lysate reagent of 50 μ l in the precipitation, mixing fully vibrates;

(4) add the described albumen extraction agent of 50 μ l, place on the vortex shaker 2 seconds of mixing;

(5) 20 ℃-25 ℃ with centrifugal 5 minutes of 13000g to discharge plasmid DNA;

(6) collect supernatant, be sure not to touch protein interface;

(7) dehydrated alcohol of adding 17 μ l plasmid DNA precipitation reagents and 250 μ l is put upside down mixing;

(8) 20 ℃-25 ℃ with centrifugal 1 minute of 13000g with the precipitation plasmid DNA;

(9) abandon supernatant, and dry plasmid DNA;

(10) with 40 μ l plasmid DNA solubilising reagents dissolving plasmid DNA ,-20 ℃ of preservations are standby.

Compared with prior art, advantage of the present invention and positively effect are: agents useful for same of the present invention adopts the N,O-Diacetylmuramidase dissolution of bacteria, removes protein and RNA by the phenol-chloroform single stage method.Extract plasmid operation time weak point with the method for extraction plasmid of the present invention, the time of carrying a kind of plasmid is no more than 10 minutes altogether; The reagent of the used extraction plasmid of the present invention is formed simple, is common experiment reagent, and wide material sources and price are low, and each component all can adopt homemade analytical pure just can extract the plasmid of high yield and purity, greatly reduces experiment fees; The plasmid DNA output height that obtains, purity is good, the pollution of no albumen and RNA, satisfy the requirement of molecular biology related experiment, the plasmid DNA of extracting can be directly used in that the enzyme in downstream is cut, connected, order-checking, protokaryon transforms and experiment such as eucaryon transfection, greatly facilitate the Bioexperiment operation, saved experimental period, improved conventional efficient.

Description of drawings

Fig. 1 uses the inventive method and uses the plasmid DNA electrophoresis contrast collection of illustrative plates that day root pillar purification kit extracts among the present invention.Among the figure, M is the dna molecular amount standard that λ-HindIII digests, and 1,2 is the plasmid DNA collection of illustrative plates that uses the inventive method to extract, and 3,4 is the identical plasmid DNA collection of illustrative plates that uses sky root pillar purification cassette to be extracted.

Embodiment

Below in conjunction with the drawings and specific embodiments technical scheme of the present invention is described in further detail.

Embodiment 1

The reagent of extraction plasmid DNA of the present invention comprises four kinds of reagent, is respectively cellular lysate reagent R1; Albumen extraction agent R2; Plasmid DNA precipitation reagent R3; Plasmid DNA solubilising reagent R4.

Described cellular lysate reagent R1:

Component: 10mM Tris-HCl (pH 8.0);

1mM EDTA；

100mM sodium-chlor;

The 5mg/ml N,O-Diacetylmuramidase.

Prepare the reagent R1 of 100ml as follows:

Get ultrapure water 75ml

0.5M?EDTA(pH8.0)?0.2ml

5M?NaCl 2ml

1M?Tris-HCl(pH?8.0)1ml

Be settled to 100ml with ultrapure water, place 121 ℃ of sterilizations 15 minutes, add the 500mg N,O-Diacetylmuramidase again, in 4 ℃ of preservations.

Described albumen extraction agent R2:

Component:

The full phenol 50ml that closes of water intaking

Chloroform 50ml

Behind the mixing, lucifuge is in 4 ℃ of preservations.

Described plasmid DNA precipitation reagent R3:

Component: 5.5M sodium-acetate+dehydrated alcohol.

Prepare earlier the 5.5M sodium-acetate of 100ml as follows:

Get sodium-acetate 45g

Ultrapure water 80ml

After the stirring and dissolving, transfer to pH7.5 with 10M NaOH, be settled to 100ml with ultrapure water, lucifuge is in room temperature preservation.

Be 1: 3 mixed with described 5.5M sodium-acetate and dehydrated alcohol according to volume ratio again, make described reagent R3.

Described plasmid DNA solubilising reagent R4:

Component: the ultrapure water of 0.1mg/ml RNase.

Prepare the reagent R4 of 100ml as follows:

Take by weighing the RNase of 10mg, add 100ml ultrapure water constant volume, 4 ℃ of preservations.

The present invention extracts plasmid DNA with above-mentioned four kinds of reagent that prepare according to following steps:

(1) shake bacterium amplification plasmid (seeing " molecular cloning experiment guide " for details) routinely, concrete steps are as follows:

A, preparation LB liquid nutrient medium and LB solid medium:

Take by weighing Tryptones 2 gram, yeast extract 1 gram, sodium-chlor 2 grams add sterile distilled water to 200 milliliter, and high pressure steam sterilization 20 minutes makes the LB liquid nutrient medium;

Take by weighing Tryptones 2 gram, yeast extract 1 gram, sodium-chlor 2 grams, agar 3 grams add sterile distilled water to 200 milliliter, and high pressure steam sterilization 20 minutes makes the LB solid medium;

The intestinal bacteria mono-clonal bacterium colony that carries plasmid of growing on B, the picking LB solid medium is inoculated in the LB liquid nutrient medium, 200 rev/mins, is placed on the 37 degree constant temperature shaking tables and shakes;

(2) get conventional 16 hours OD of cultivation ₆₀₀Reach the good bacterium bacterium liquid 1.5ml of growth conditions more than 0.6 in the 1.5ml centrifuge tube, under the room temperature centrifugal 30 seconds, abandon supernatant with 13000g;

(3) add 50 μ l reagent R1 in precipitation, mixing fully vibrates;

(4) add 50 μ l reagent R2 again, 2 seconds of mixing on the vortex shaker;

(5) under the room temperature with centrifugal 5 minutes of 13000g to discharge plasmid DNA;

(6) draw supernatant with aseptic liquid-transfering gun, be sure not to touch protein interface;

(7) in supernatant, add the reagent R3 of 17 μ l and the dehydrated alcohol of 250 μ l, put upside down mixing;

(8) under the room temperature with centrifugal 1 minute of 13000g with the precipitation plasmid DNA;

(9) abandon supernatant, and dry plasmid DNA;

(10) with the reagent R4 dissolving plasmid DNA of 40 μ l ,-20 ℃ of preservations are standby.

The plasmid DNA of being extracted can be verified with 1% agarose gel electrophoresis.As shown in Figure 1, the thalline that contains same plasmid DNA is used the inventive method and is used the plasmid DNA electrophoresis contrast collection of illustrative plates that day root pillar purification kit extracts.Among Fig. 1, M is the DL2000 molecular weight standard, and 1,2 is the plasmid DNA of using the inventive method to extract, and 3,4 is the corresponding plasmid DNA of using sky root pillar purification cassette to be extracted.Use the plasmid DNA of two kinds of test kit gained as seen from the figure, electrophoretic band is clear, and no RNA pollutes.Illustrate and use the present invention plasmid DNa that extracts and the same plasmid DNA of using sky root pillar purification kit to extract relatively, on purity and output, there is not obviously difference, but it is a lot of that the reagent of extraction plasmid DNA of the present invention reduces with respect to sky root pillar purification kit cost, and simple to operate, operate used time weak point.

Above embodiment is only in order to illustrating technical scheme of the present invention, but not limits it; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.

Claims (3)

1. a reagent that extracts plasmid DNA is characterized in that it comprises cellular lysate reagent, albumen extraction agent, plasmid DNA precipitation reagent and plasmid DNA solubilising reagent; The component of described cellular lysate reagent is: 10mM Tris-HCl, 1mM EDTA, 100mM sodium-chlor and 5mg/ml N,O-Diacetylmuramidase are solvent with the ultrapure water, and the pH value of described Tris-HCl is 7.4-8.0; The component of described albumen extraction agent is: volume ratio is 1: 1 phenol and a chloroform mixture; The component of described plasmid DNA precipitation reagent is: 5.5M sodium acetate soln and the dehydrated alcohol of PH7.5, and described sodium acetate soln and dehydrated alcohol volume ratio are 1: 3; The component of described plasmid DNA solubilising reagent is the ultrapure water that contains 0.1mg/ml RNase.

2. a kind of reagent that extracts plasmid DNA according to claim 1, the best pH value that it is characterized in that described reagent Tris-HCl is 8.0.

3. a reagent according to claim 1 extracts the method for plasmid DNA, it is characterized in that it may further comprise the steps:

(1) shakes bacterium amplification plasmid; The intestinal bacteria of carrying plasmid are inoculated in the LB solid medium, select the mono-clonal colony inoculation, be placed on the 30-38 ℃ of constant temperature shaking table with 150-250 rev/min of wave and culture in LB liquid nutrient medium amplification cultivation;

(2) get 12-16 hour OD of cultivation ₆₀₀Reach the good bacterium bacterium liquid 1.5ml of growth conditions more than 0.6 in the 1.5ml centrifuge tube, under 20 ℃-25 ℃ centrifugal 30 seconds, abandon supernatant with 13000g;

(3) add the described cellular lysate reagent of 50 μ l in the precipitation, mixing fully vibrates;

(4) add the described albumen extraction agent of 50 μ l, place on the vortex shaker 2 seconds of mixing;

(5) 20 ℃-25 ℃ with centrifugal 5 minutes of 13000g to discharge plasmid DNA;

(6) collect supernatant, be sure not to touch protein interface;

(7) dehydrated alcohol of adding 17 μ l plasmid DNA precipitation reagents and 250 μ l is put upside down mixing;

(8) 20 ℃-25 ℃ with centrifugal 1 minute of 13000g with the precipitation plasmid DNA;

(9) abandon supernatant, and dry plasmid DNA;

(10) with 40 μ l plasmid DNA solubilising reagents dissolving plasmid DNA ,-20 ℃ of preservations are standby.

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