Method for extracting plasmids with endotoxin removed on large scale

A large-scale technology of endotoxin, applied in the direction of biochemical equipment and methods, microbial measurement/testing, DNA preparation, etc., can solve the problems of endotoxin removal, high cost, high temperature control requirements, etc., and achieve easy control and removal Pollution, the effect of simple operation methods

Active Publication Date: 2017-12-22
上海埃秀马生物科技有限公司
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Problems solved by technology

However, this process cannot remove endotoxin. Endotoxin is a component of the cell wall of Gram-negative bacteria and is a heat source substance. Once injected into the animal body, endotoxin-containing substances can cause fever, microcirculation disturbance, and endotoxin shock. and disseminated intravascular coagulation and other toxic effects; in molecular biology experiments, the presence of endotoxin will reduce the transfection efficiency of plasmids and the expression efficiency of cells
[0005] The boiling cracking method requires high temperature control, which makes the boiling method less stable in laboratory preparation, and the contamination is not easy to remove, so it is not suitable for large-scale plasmid extraction
[0006] The kit method is fast but expensive, not suitable for large-scale plasmid extraction

Method used

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Embodiment 1

[0029] A method for extracting endotoxin-removed plasmids on a large scale, comprising the following steps:

[0030] Step S1, carrying out bacterial culture, performing cell resuspension, and obtaining a bacterial culture containing plasmid DNA;

[0031] Step S2, adding an appropriate amount of NaOH-SDS solution for mixing, centrifuging to obtain a supernatant containing the roughly separated plasmid DNA, filtering the supernatant with filter paper, and transferring the supernatant into a centrifuge tube;

[0032] Step S3, add isopropanol to the centrifuge tube to precipitate the plasmid, centrifuge and discard the supernatant; add TE solution to dissolve the precipitate, add NH 4 After the Ac solution, centrifuge to retain the supernatant; add absolute ethanol to the supernatant to re-precipitate to obtain relatively purified plasmid DNA, and add TE solution to dissolve the precipitate;

[0033] Step F, adding RNase A to remove RNA;

[0034] Step S4, first add NaCl; then ad...

Embodiment 2

[0039] This embodiment provides a method for large-scale extraction of endotoxin-removed lentiviral plasmids, which specifically includes the following operations:

[0040] Bacterial culture: Pick a single clone colony transformed with the pLVX-ZsGreen lentiviral plasmid from the LB plate containing Amp resistance and inoculate it in 5 mL of LB culture medium containing Amp resistance, and cultivate overnight at 37°C on a constant temperature shaker (250rpm). Take 1 mL of the overnight culture solution and inoculate it into 350 mL of the LB culture solution with a final concentration of Amp of 50 μg / mL, and incubate overnight on a constant temperature shaker at 37°C with the rotation speed set at 250 rpm. All the overnight culture solution was poured into a 500mL centrifuge bottle, and centrifuged at 4,000rpm for 10min at 4°C.

[0041] Resuspend the cells: Discard the supernatant as completely as possible, break up the cells with a shaker, and gradually add 5ml TE solution (fi...

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Abstract

The invention relates to a method for extracting plasmids with endotoxin removed on a large scale. The method comprises the steps of acquiring roughly-separated plasmid DNA by virtue of a classical alkaline lysis method, sequentially adding isopropanol, an NH4Ac solution, RNase A and NaCl, adding a mixed solution of PEG6000 and NaCl, dissolving and precipitating by virtue of a TE solution, repeatedly precipitating by virtue of absolute ethyl alcohol, washing and precipitating by virtue of 70% alcohol, simultaneously coordinating with centrifugation, drying, and finally dissolving and precipitating by virtue of the TE solution, so as to obtain target plasmids. According to the method, the pollution caused by endotoxin to plasmid DNA can be well removed, all reagents are standing chemical reagents in a laboratory, and the operation method is simple, easy to control and suitable for large-scale extraction of plasmids. The concentration of the plasmids can reach 5mg/mL, the endotoxin is lower than 5EU/mu g, and the plasmids can be applied to digestion, DNA sequencing, cell transfection, virus packaging and clinic animal immunization experiments.

Description

technical field [0001] The invention relates to a method for extracting plasmids, in particular to a method for extracting endotoxin-removed plasmids on a large scale. Background technique [0002] Plasmid is an extrachromosomal genetic unit capable of autonomous replication, a carrier that can introduce foreign genes into bacteria for amplification and expression, and a major tool for genetic engineering. To use plasmids as carrier molecules for gene cloning, an important condition is to obtain large quantities of purified plasmid DNA molecules. Studies have shown that high-quality plasmids are the basis for success in cell transfection, nucleic acid vaccines, and gene therapy. With the rapid development of the research and application of genetically recombinant drugs, the demand for recombinant plasmids continues to increase, and conventional laboratory preparation methods can no longer meet the requirements well. Therefore, it is important to explore a reliable large-sca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2521/327
Inventor 陈碧花易丹王国胜黄俊
Owner 上海埃秀马生物科技有限公司
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