Method for purifying plasmids

A plasmid, calcium chloride solution technology, applied in DNA preparation, recombinant DNA technology and other directions, can solve the problems of ineffective removal of pollutants, increase removal costs, etc., and achieve the effects of removing RNA pollution, simple operation, and cost reduction

Pending Publication Date: 2022-03-11
NANJING BIOHENG BIOTECH CO LTD
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Problems solved by technology

However, some methods commonly used at present cannot effectively remove these pollutants, especially RN

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  • Method for purifying plasmids
  • Method for purifying plasmids

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Embodiment 1

[0017] Embodiment 1. Purify plasmid with the method of the present invention

[0018] 1. Alkaline lysis of E. coli culture: resuspend the E. coli culture containing the plasmid to be purified evenly with resuspension buffer (TE buffer, pH8.0), and then add an equal proportion of lysate (0.2M NaOH+2 % SDS solution), after 5 minutes, add an equal proportion of 3M potassium acetate buffer to neutralize to obtain 3L lysate;

[0019] 2. Divide the lysate into 5 parts, 600ml each, add 125ml of ultrapure water to one of them (that is, the concentration of calcium chloride is 0, sample 1), and add 125ml of various concentrations of CaCl 2 Solution (pH5.0-7.0), so that the final calcium chloride concentration in the system is 0.1M (sample 2), 0.3M (sample 3), 0.5M (sample 4), 1M (sample 5).

[0020] 3. After standing at room temperature for 30 minutes, take 10 μl of the supernatant for electrophoresis (for electrophoresis results, see figure 1 1-5 in ), and take 400ml supernatant, c...

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Abstract

The invention relates to a plasmid purification method for industrial production, which comprises the following steps: (1) splitting a bacterial culture containing plasmids to be purified to obtain a lysate; (2) adding a calcium chloride solution with the final concentration of 0.5-1M into the lysate to obtain a crude extract; (3) filtering the crude extract by using a membrane filter to obtain filtrate; and (4) carrying out chromatography on the filtrate by using an anion exchange column to obtain purified plasmids.

Description

technical field [0001] The present invention describes a method for purifying plasmids. This method is mainly used for plasmid preparation in pilot scale and industrial production in the field of biopharmaceuticals. Background technique [0002] Plasmid is an extrachromosomal genetic element widely present in nature. As a carrier of genetic information, plasmids have been widely used in gene transfer operations. Especially in recent years, with the rapid development of advanced therapeutic methods (including gene therapy and cell therapy), the production of plasmids has gradually shifted from laboratory to industrial mass production. [0003] In the industrialized large-scale production of plasmids, many challenges are faced, including improving the purity of plasmids and obtaining pure plasmids that meet pharmaceutical requirements. According to the requirements of current regulations, the final product of plasmids should remove as much pollutants as possible, such as ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1017C12N15/101
Inventor 李彦林陈明曦任江涛贺小宏王延宾韩露
Owner NANJING BIOHENG BIOTECH CO LTD
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