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Simple nucleic acid purifying method

A simple and nucleic acid technology, applied in the field of molecular biology, can solve the problems of unfavorable scientific research and routine medical testing, high price, unfavorable health of environmental protection operators, etc., and achieve the effect of reducing experimental cost, simple production and easy mastery

Inactive Publication Date: 2011-04-27
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In summary, conventional nucleic acid extraction methods and commercialized nucleic acid extraction kits still have the following problems: First, the price is relatively expensive, which is not conducive to scientific research and routine medical testing
Second, toxic substances such as phenol and chloroform need to be used, which is not conducive to environmental protection and the health of operators

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0049] Simple purification method of plant genomic DNA:

[0050] (1) Weigh 0.3g of plant material of planegrass blade, cut it into pieces, put it in a mortar, add 1.5ml of CTAB lysate or SDS lysate for grinding, wherein CTAB lysate contains: CTAB 2%, sodium chloride 1.4mol / L , EDTA 0.02mol / L and Tris0.1mol / L, the pH value is 8.0; SDS lysate contains: SDS 4%, sodium chloride 0.5mol / L, EDTA 0.05mol / L, and Tris 0.2mol / L, pH The value is 7.2.

[0051] (2) Transfer the grinding solution to a 2ml centrifuge tube and place in a water bath at 65°C for 60min.

[0052] (3) Add an equal volume of adsorption-promoting solution sodium chloride 5 mol / L, mix well, centrifuge at 12000 rpm for 5 min, and take the supernatant.

[0053] (4) Transfer the supernatant to the prepared simple centrifugal purification column, let it stand for 2 minutes, centrifuge at 8000rpm for 1 minute, pour off the liquid in the collection tube, and repeat this step until all the supernatant obtained in step (3) is...

Embodiment 2

[0061] Simple purification method of E. coli plasmid DNA:

[0062] (1) Take 1.5 ml of Escherichia coli JM109 culture containing PBI121 plasmid and centrifuge at 12000 rpm for 1 min. Aspirate and discard the culture medium, leaving the bacterial pellet as dry as possible.

[0063] (2) Resuspend the obtained bacterial pellet in 100 μl of solution I with a pH value of 8.0 composed of reagents and volume concentrations: Tris 25 mmol / L, EDTA 10 mmol / L and glucose 50 mmol / L, and shake vigorously.

[0064] (3) Add 200 μl of reagent and volume concentration: 0.2 mol / L sodium hydroxide and 1% (W / V) solution II to the solution II, cover the tube mouth tightly, quickly invert the centrifuge tube 5 times, do not use force in this step Shake, mix well, and place at room temperature for 5 minutes.

[0065] (4) Add 200 μl of solution III consisting of reagent and volume concentration: potassium acetate 3mol / L and glacial acetic acid 11.5% (V / V), cap the tube tightly, turn the tube upside d...

Embodiment 3

[0075] A simple purification method for extracting plant total RNA by lysing cells with guanidine isothiocyanate:

[0076] (1) Cut 0.1g of the planegrass blade plant material into pieces and put it in a mortar, add 1ml of reagent and volume concentration of guanidine isothiocyanate 6mol / L, sodium lauryl sarcosine 0.83% (W / V) The guanidine isothiocyanate lysate composed of sodium citrate 42mmol / L was ground.

[0077] (2) Transfer 0.5ml of grinding fluid to a 1.5ml centrifuge tube, add 100μl of 3mol / L sodium acetate buffer, adjust the pH to 4.0 with glacial acetic acid, mix well, and place on ice for 5min.

[0078] (3) Add 850 μl of DEPC-treated water, mix well, centrifuge at 12000 rpm for 5 min, and take the supernatant.

[0079] (4) Add 0.5 times the volume of absolute ethanol to the supernatant, and mix well.

[0080] (5) Transfer the obtained solution to a simple purification column, let it stand for 2 minutes, centrifuge at 8000 rpm for 1 minute, pour off the liquid in th...

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Abstract

The invention discloses a simple nucleic acid simplifying method which comprises processes of cracking, adsorption promotion, washing and elution. The method is characterized by comprising preparation methods and use methods of a simple purifying column, cracking liquid, adsorption promoting liquid and washing liquid special for nucleic acid purification, wherein the simple purifying column is a 1.5 ml centrifuge tube with a hole on the bottom; a small mass of absorbent cotton is compacted on the tube bottom; 0.3-1.0 g of glass powder with diameter of 120-130 mum is added as a purifying column body; an uncovered 2.0 ml centrifuge tube is sleeved outside the column body to serve as a collection tube, therefore, the collection tube is easily manufactured by using a common EP (Eppendorf) tube, cotton and glass powder. With a wide application range, the method is directly applied to the extraction and purification of genome DNA, plasmid DNA and total RNA, as well as the recycling, centrifugation and purification of PCR (polymerase chain reaction) products; the operation steps are concise and easy to master, and the requirements on the experiment operation technique are not high; moreover, the method has the advantages of high purification purity, high integrity, short purification time and low experiment cost; and as toxic substances such as phenol and chloroform are not used in the purification process, the method is environmentally friendly and energy-saving.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a simple method for purifying nucleic acid. Background technique [0002] With the development of molecular biology, nucleic acid extraction and purification has become an essential work in molecular biology research. The first step in experimental operations such as disease diagnosis, cloning, sequencing, amplification, hybridization, and analysis is to extract complete, high-quality DNA nucleic acid. To this end, people have developed a variety of nucleic acid purification methods, mainly including phenol-chloroform extraction, media adsorption and other methods. Among them, the phenol-chloroform extraction method is the most classic traditional method. This method involves multiple centrifugation operations in the process of use, which takes a long time, consumes a lot of consumables, is complicated and cumbersome, and inevitably uses volatile and toxic chemical reagen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 何海旺何龙飞李创珍
Owner GUANGXI UNIV
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