Batch extraction method for plasmid by alkaline lysis

A technique of extracting plasmids and alkaline lysis, which is applied in the field of biochemical experiments, can solve the problems of difficult DNA renaturation of the main chromosome, and achieve the effects of low cost, simple operation, and low requirements for experimental conditions

Inactive Publication Date: 2014-03-05
辛竹
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Problems solved by technology

Due to the plasmid and the main chromosome Topology Different, although the two chains of the former are separated during denaturation, they are still intertwined and not separated; but the latter is completely denatured and even breaks. Therefore, when adding acidic pH4.8 Potassium acetate When the pH value of the solution is lowered to restore the pH value of the solution to a lower near-neutral level, the two small molecule single strands of the plasmid can quickly anneal to restore the double-stranded structure, but the main chromosome DNA is difficult to anneal

Method used

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Embodiment Construction

[0008] A method for extracting a large number of plasmids by alkaline lysis, the steps are as follows: firstly prepare appropriate Chlamydomonas; transfer its bacterial solution to a high-temperature sterilized centrifuge tube; discard the supernatant; add preheated solution to the bacterial cell, Vortex and shake evenly; let stand for 5-8 minutes; use the upper part to filter the precipitate into a new centrifuge tube; add 2-2.5 times the mentioned monoethanolamine; mix well and let stand again; wash with ethanol, Precipitation; dry at room temperature. The Chlamydomonas must have reached the middle and late stages of growth, and 24 hours before use, it needs to be placed in a centrifuge tube, centrifuged at a high speed, and the supernatant is taken, coated on an agar plate, and kept under the condition of 23 degrees Celsius of light for continuous 48 hours; the plasmid obtained after drying needs to be added with isopropanol, and the mass fraction is 65%.

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Abstract

The invention discloses a batch extraction method for plasmid by alkaline lysis. The method comprises the following steps: preparing proper chlamydomonas, transferring bacteria liquid in a centrifuge tube disinfected at high temperature; abandoning a supernatant; adding a preheating solution in thalline, performing vortex oscillation, uniformly shaking; standing for 5-8 minutes; filtering the deposition to a new centrifuge tube; adding ethanolamine with volume of 2-2.5 times; uniformly mixing and standing again, washing by ethanol and depositing; and air drying at room temperature. The growth of chlamydomonas is required at the middle and later periods, and the chlamydomonas is placed in the centrifuge tube 24 hours before usage, high speed centrifugation running is carried out, an supernatant is taken to film to an agar plate, the continuous illumination at 23 DEG C lasts for 48 hours; isopropanol is required to be added in the plasmid after air drying, wherein the mass fraction is 65%. The method has the beneficial effects of simple operation, low cost and low experiment condition requirement, can be repeatedly operated to obtain the expectation effect.

Description

technical field [0001] The invention relates to the field of biochemical experiments, in particular to a method for extracting a large number of plasmids by alkali lysis. Background technique [0002] Alkaline lysis is the most common and effective method for extracting plasmids. It is based on the difference in denaturation and renaturation between chromosomal DNA and plasmid DNA to achieve the purpose of separation. The principle of its use is: high pH denatures plasmid DNA and chromosomal DNA, and precipitates proteins at the same time. Then adjust the pH value to neutral, the plasmid DNA is small, and it is easy to anneal into double strands. Chromosomal DNA is relatively large, will not refold, and will tangle into a network of insoluble matter, which can be removed by centrifugation. Alkaline lysis is the more commonly used extraction method. The advantage is that the harvest rate is high, it is suitable for most bacterial strains, and the obtained product can meet ...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N15/10C12R1/89
Inventor辛竹
Owner辛竹