Method for extracting plasmids with endotoxin removed on large scale

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...

AI Technical Summary

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