CN101717421A - Method for regenerating nucleic acid purification column - Google Patents

Abstract

The invention discloses a method for regenerating a nucleic acid purification column, which has simple operation and strong application and convenient generalization. The method comprises the following steps of: separating the used nucleic acid purification column from an external sleeve, placing the nucleic acid purification column and the external sleeve into 3.68MH2SO4 to boil for 10 min, washing the purification column and the sleeve for three times by using sterile water; assembling the purification column and the sleeve, adding 700 microliter of sterile water to the purification column, centrifuging for 1 min at 12000*g, discarding filtrate, repeating the process twice; adding 40 microliter of sterilized Tris-HCL with the length of 2.5mM and the pH of 8.5 to the center of a silica gel film, standing for 1 min at the room temperature, centrifuging for 2 min at 12000*g, sterilizing the purification column and the external sleeve with high pressure for 15 min at 121 DEG C, and drying at 60 DEG C for later use.

Description

The method of regenerating nucleic acid purification column

Technical field:

The present invention relates to a kind of method of regenerating nucleic acid purification column, the method for the regenerating nucleic acid purification column that especially a kind of simple to operate, applicability is strong, be convenient to popularize.

Background technology:

Nucleic acid purification is one of routine techniques of molecular biology research.Existing nucleic acid purification method is divided into following three kinds usually: the imitative extraction process of classical alkaline lysis and phenol; Gai Liang pellosil absorption method not; Commodity purification column method.Because classical way time-consuming and the limitation that does not improve the pellosil absorption method make that simple, fast commodity purification column purification process has obtained using widely.Yet commercialization nucleic acid purification post price is higher and only use as medical disposable material, has not only increased the experiment expense, and the depleted purification column also can pollute environment, has potential hazardness.Therefore, Chinese scholars begins to seek effective regenerating nucleic acid purification column method.At present, though existing report about regenerating nucleic acid purification column research, because of its complicated operation, practical application is undesirable, be not easy to popularize etc., uses the pollution of disposable nucleic acid purification post and waste problem still to can not get effective solution.

Summary of the invention:

The present invention is in order to solve the above-mentioned technical problem of existing in prior technology, provide a kind of simple to operate, applicability strong, the method for the regenerating nucleic acid purification column being convenient to popularize.

Technical solution of the present invention is: a kind of method of regenerating nucleic acid purification column is characterized in that carrying out as follows:

A. used nucleic acid purification post is separated with outer sleeve, all place 3.68M H ₂SO ₄In boil 10min, again with aseptic water washing purification column and sleeve pipe 3 times;

B. assemble purification column and sleeve pipe, add 700 μ l sterilized waters in purification column, the centrifugal 1min of 12000 * g abandons filtrate, repeats twice of this process;

C. the 2.5mM that adds 40 μ l sterilization to pellosil central authorities, the Tris-HCL of pH8.5, room temperature leaves standstill 1min, the centrifugal 2min of 12000 * g;

D. purification column and outer tube be through 121 ℃ of autoclaving 15min, and 60 ℃ of dryings are stand-by.

Thereby the present invention has made full use of nucleic acid pellosil isolating principle on hydrolysis and the nucleic acid purification post under acidic conditions, reaches the purpose of eliminating residual pollution of nucleic acid on the purification column.Used hydrolysing agent is certain density analytical pure sulfuric acid solution, and Tris-HCL is the conventional reagent in laboratory, in addition need not other any chemical reagent, has reduced the running cost of regeneration, has reduced the pollution to environment; Therefore the operation whole process only needed about 20 minutes, and other renovation process of having reported more is quick, has significantly improved working efficiency, had stronger applicability, advantage such as was convenient to popularize.Detect and estimate by PCR, restriction enzyme digestion and nucleic acid colorimetry quality, find no the nucleic acid residual contamination, and can continue to use the regeneration purification column.Detected result shows, through this technology regenerated purification column reproducible utilization 6 times at least.

Description of drawings:

Fig. 1 is the PCR detection figure of purification column filtrate after the embodiment of the invention manipulation of regeneration.

Fig. 2 is that embodiment of the invention regeneration nucleic acid purification column detects content and the quality figure that actifier column extracts plasmid DNA with ultraviolet spectrometry colorimetry (A) and electrophoresis (B).

Fig. 3 is that embodiment of the invention regeneration nucleic acid purification column detects the quality figure that actifier column extracts plasmid DNA with PCR.

Fig. 4 is that embodiment of the invention regeneration nucleic acid purification column detects the quality figure that actifier column extracts plasmid DNA with restriction enzyme digestion.

Embodiment:

A. used nucleic acid purification post is separated with outer sleeve, all place 3.68M H ₂SO ₄In boil 10min, again with aseptic water washing purification column and sleeve pipe 3 times;

B. assemble purification column and sleeve pipe, add 700 μ l sterilized waters in purification column, the centrifugal 1min of 12000 * g abandons filtrate, repeats twice of this process;

C. the 2.5mM that adds 40 μ l sterilization to pellosil central authorities, the Tris-HCL of pH8.5, room temperature leaves standstill 1min, the centrifugal 2min of 12000 * g;

D. purification column and outer tube be through 121 ℃ of autoclaving 15min, 60 ℃ of dryings, stand-by getting final product.

As follows to regenerate filtrate and the quality examination of regeneration nucleic acid purification column that nucleic acid purification column process produced of the present invention:

1. the filtrate that produces in the b step is carried out PCR and detect, reaction system is as follows:

Template (filtrate) 1 μ l,

EX?Buffer(Mg ²⁺Plus)2.5μl，

dNTP?Mixture(each?2.5mM)2.0μl，

Each 0.5 μ l (10pmol/ μ l) of forward primer and reverse primer,

EX Taq 0.14 μ l mends to 25 μ l with water.

Reaction conditions is: 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 2min.After reaction finishes, respectively get 5 μ l amplified productions and carry out the detection of 1% agarose gel electrophoresis.The result is as shown in Figure 1 following.R1-R6 represents the number of times of manipulation of regeneration respectively among the figure, and M is Maker; Get 3 posts at random after each manipulation of regeneration and detect, below all with.

2. regenerating nucleic acid purification column utilizes the detection of effect

(1) gel electrophoresis and spectrophotometer colorimetric assay detect

Criticize the plasmid that is connected with exogenous dna fragment with new purification column together with the purification column extraction through different regeneration times, the plasmid DNA of being extracted is carried out electrophoresis detection in 1% sepharose.Simultaneously, adopt ultraviolet spectrophotometer that corresponding plasmid DNA is carried out detection by quantitative, the result as shown in Figure 2, New represents new post among the figure, below all with.

(2) pcr amplification detects

The plasmid of getting the extraction of new post and actifier column respectively carries out PCR and detects, and reaction system and reaction conditions carry out PCR detection (circulation is 30 times) with above-mentioned to the filtrate that produces in the b step, and the result as shown in Figure 3.

(3) restricted double digestion detects

The plasmid of getting the extraction of new post and actifier column respectively carries out restricted double digestion reaction.Reaction system comprises 10 μ l plasmids, 4 μ l, 10 * Buffer, and HindIII 1 μ l, BamHI1 μ l mends to 20 μ l with water.37℃，1h。Enzyme is cut product carry out the detection of 1% agarose gel electrophoresis, the result as shown in Figure 4.

Claims (1)

1. the method for a regenerating nucleic acid purification column is characterized in that carrying out as follows:

A. used nucleic acid purification post is separated with outer sleeve, all place 3.68M H ₂SO ₄In boil 10min, again with aseptic water washing purification column and sleeve pipe 3 times;

B. assemble purification column and sleeve pipe, add 700 μ l sterilized waters in purification column, the centrifugal 1min of 12000 * g abandons filtrate, repeats twice of this process;

C. the 2.5mM that adds 40 μ l sterilization to pellosil central authorities, the Tris-HCL of pH8.5, room temperature leaves standstill 1min, the centrifugal 2min of 12000 * g;

D. purification column and outer tube be through 121 ℃ of autoclaving 15min, and 60 ℃ of dryings are stand-by.

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