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Preparation method of plasmid DNA

A plasmid and mixing technology, applied in DNA preparation, recombinant DNA technology, etc.

Inactive Publication Date: 2016-03-02
刘铮
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is not as fast as the QIAGEN kit, it is still worth promoting due to the abundant and cheap domestic human resources

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0031] The present invention includes the following steps.

[0032] 1) Pick a single colony containing the plasmid and inoculate it into the LB medium with corresponding resistance, and culture it with shaking overnight. Take the overnight cultured bacterial liquid, inoculate it into the LB medium with corresponding resistance, and culture it with shaking.

[0033] 2) Transfer the bacterial solution to a centrifuge bottle, centrifuge, and remove the supernatant.

[0034] 3) Resuspend the bacterial pellet in Solution I and shake vigorously to disperse and mix the bacterial cells evenly.

[0035] 4) Add solution II, invert the centrifuge bottle gently several times to mix well, and place the centrifuge bottle on ice to lyse the bacteria.

[0036] 5) Add the pre-cooled solution Ⅲ, gently invert the centrifuge bottle several times to mix well, and place it on ice. Centrifuge for 10 min.

[0037] 6) Pour the supernatant into a centrifuge tube, add isopropanol to mix, centrifuge,...

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PUM

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Abstract

The invention belongs to the technical field of preparation methods and particularly relates to a preparation method of plasmid DNA. The preparation method of high-purity plasmid DNA is provided. The preparation method includes the following steps that firstly, single colonies containing plasmids are selected and inoculated to an LB culture medium with the corresponding resistance, oscillation culture is performed overnight, bacterial liquid which is cultured overnight is taken and is inoculated to an LB culture medium with the corresponding resistance, and oscillation culture is performed; secondly, the bacterial liquid is transferred into a centrifugal bottle, centrifuging is performed, and supernatant liquid is removed; thirdly, bacteria precipitate in a resuspensed solution I, violent oscillation is performed, and thalli are uniformly scattered; fourthly, a solution II is added, the centrifugal bottle is reversed many times gently so that the solutions can be uniformly mixed, the centrifugal bottle is placed on ice, and the bacteria are decomposed; fifthly, a pre-cooled solution III is added, the centrifugal bottle is reversed many times gently so that the solutions can be uniformly mixed, the centrifugal bottle is placed on ice, and centrifuging is performed for 10 min.

Description

technical field [0001] The invention belongs to the technical field of DNA preparation methods, in particular to a method for preparing plasmid DNA. Background technique [0002] High-quality DNA is essential for efficient transfection. Previous researchers used DNA obtained by CsCl gradient centrifugation for transfection. However, this method requires expensive ultracentrifuges and is time-consuming and laborious. Currently, few people use it. At present, the purification kits based on column chromatography are mostly used in the extraction of ultrapure plasmids, and the products of QIAGEN Company in Germany are the most famous. QIAGEN uses patent-based anion exchange chromatography technology to obtain 20 μg-10 mg of plasmid DNA equivalent to 2 times of CsCl ultracentrifugation within 70 minutes, but the price of this kit is beyond the reach of most domestic laboratories. Said it was unbearable. Saporito-Irwin SM et al. [7] A method for manual extraction of plasmids wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 刘铮
Owner 刘铮