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CN-121975797-A - Enhanced lysate for extracting plasmid DNA and application thereof

CN121975797ACN 121975797 ACN121975797 ACN 121975797ACN-121975797-A

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses an enhanced lysate for plasmid DNA extraction and application thereof. The enhanced pyrolysis liquid comprises 0.1-0.3mol/L sodium hydroxide, 0.5-2% sodium dodecyl sulfate, 0.1-0.5% quaternary ammonium salt and the like. According to the invention, by improving the cracking efficiency of the cracking liquid under unit volume, the processing range of the bacterial load can be greatly improved under the same reaction liquid volume, the bacterial cracking capacity of 100-500ml bacterial load can be effectively compatible, the bacterial limitation that only 100-200ml bacterial load can be extracted under the same reaction liquid volume of the traditional cracking liquid is improved, the problem that the cracking cannot be fully performed due to the too high viscosity of the high bacterial load is solved, the dosage of pretreatment reagent required by extraction is reduced, the volume of the post-centrifugation column is reduced, the operation steps are reduced, and the experimental operation time is saved.

Inventors

  • MA LIE
  • ZOU AIHONG
  • MA JIANGTAO
  • SONG XINWEN
  • GENG LIANG
  • XIN WEN

Assignees

  • 北京全式金生物技术股份有限公司

Dates

Publication Date
20260505
Application Date
20260212

Claims (10)

  1. 1. An enhanced lysate for plasmid DNA extraction, characterized in that the enhanced lysate comprises 0.1-0.3mol/L sodium hydroxide, 0.5-2% sodium dodecyl sulfate and 0.1-0.5% quaternary ammonium salt; wherein the quaternary ammonium salt is selected from one or more of benzalkonium bromide, polyhydroxy propyl dimethyl ammonium chloride, dimethyl diallyl ammonium chloride, bisdodecyl dimethyl ammonium chloride and octadecyl dimethyl benzyl ammonium chloride.
  2. 2. The enhanced cleavage liquid of claim 1 further comprising 0.1 to 1mol/L sodium succinate.
  3. 3. The enhanced cleavage liquid according to claim 2, wherein the enhanced cleavage liquid comprises 0.2mol/L sodium hydroxide, 1% sodium dodecyl sulfate, 0.5 mol/L sodium succinate and 0.2% quaternary ammonium salt.
  4. 4. A method of plasmid DNA extraction comprising: (1) Adding the heavy suspension into a bacterial sample which is obtained by centrifuging and enriching 100-500ml of bacterial liquid, and re-suspending and scattering bacterial bodies; (2) Adding the enhanced lysate of any one of claims 1-3 into the resuspended bacterial liquid of step (1), gently reversing and mixing to obtain a lysate; (3) Adding the neutralization solution into the cracking product, and gently mixing until egg-shaped aggregation is formed; (4) Centrifuging, transferring the supernatant to a new centrifuge tube, adding an endotoxin-removing reagent and a proper amount of isopropanol, and uniformly mixing; (5) Adding all the liquid in the step (4) into a silicon matrix column, and centrifuging to remove waste liquid; (6) Adding rinsing liquid into the silicon matrix column, and centrifuging to remove waste liquid; (7) Emptying the centrifugal column, and discarding residual liquid; (8) And adding a proper amount of preheated eluent into the silicon matrix column, and centrifuging to obtain plasmid DNA.
  5. 5. The method of claim 4, wherein the heavy suspension is added in an amount of 10-13ml, the enhanced lysate is added in an amount of 10-13ml, and the neutralization solution is added in an amount of 10-13ml; preferably, in the step (2), the cleavage is carried out for 3 to 5 minutes to obtain a cleavage product.
  6. 6. The method according to claim 4, wherein in the step (4), the centrifugation condition is 8,000 to 10,000g for 10 to 15min, and in the steps (5), (6), (7) and (8), the centrifugation condition is 8,000 to 10,000g for 1 to 3min.
  7. 7. Use of an enhanced lysate for plasmid DNA extraction according to any one of claims 1-3 for the extraction of plasmid DNA.
  8. 8. Use of an enhanced lysate for plasmid DNA extraction according to any one of claims 1-3 for the preparation of a product for plasmid DNA extraction.
  9. 9. An ultra-large amount plasmid DNA extraction kit comprising the enhanced lysate for plasmid DNA extraction of any one of claims 1-3.
  10. 10. The method of claim 9, further comprising the step of resuspension, neutralization, rinsing and/or eluting.

Description

Enhanced lysate for extracting plasmid DNA and application thereof Technical Field The invention belongs to the technical field of molecular biology, and particularly relates to an enhanced lysate for plasmid DNA extraction and application thereof. Background Plasmid DNA is used as a key tool in genetic engineering, plays a role of a bridge tie between a molecular biological experiment and a cell biological experiment, and is often used as a carrier to carry exogenous genes into a receptor cell or a cell-free system so as to realize the replication, transcription and expression of genes due to the functional characteristics of easy editing, easy preparation, shuttle expression and the like. In addition to conventional scientific research applications, plasmid DNA is also used in the development and production of recombinant proteins, nucleic acid vaccines, and other biomedical materials. The extraction method of plasmid DNA mainly comprises boiling method and alkaline lysis method. Wherein, the boiling method is to destroy the thallus structure by lysozyme, then separate the plasmid DNA from insoluble impurities by boiling treatment, and finally obtain the plasmid DNA by ethanol precipitation and centrifugal operation. The method has the disadvantages of limited single treatment capacity, high cost, long time consumption, difficult control of extraction quality and the like, and cannot meet the current requirement for rapidly preparing a large amount of plasmids. The current mainstream technique for commercial preparation of plasmid DNA is alkaline lysis. The method is generally matched with a silicon matrix column purification technology, so that the simplification of a plasmid extraction process and the improvement of plasmid purity and yield are realized. However, the existing commercialized method for processing a bacterial sample with a bacterial liquid volume of 100-500ml cannot complete extraction under the same system, and especially, the extraction system is huge due to the increased reagent volume along with the increase of the bacterial liquid volume, so that the operation difficulty is increased by replacing a large-volume container, and the centrifugation times and the extraction time are increased. This not only reduces the experience of plasmid preparation, but also limits the use scenarios of the product. Therefore, there is a need to provide a lysis system for plasmid DNA mass production which is small in amount and compatible with a bacterial sample of 100-500ml bacterial liquid amount, so as to solve the above-mentioned problems. Disclosure of Invention Aiming at the problems of the prior art, the invention aims to provide an enhanced lysate which is applicable to the preparation of a large quantity of plasmid DNA to a very large quantity of plasmid DNA and application thereof. The sample cracking capacity of the cracking liquid is improved, the consumption of reagents is reduced, and further tedious centrifugation steps are reduced, so that the extraction efficiency is improved. In order to achieve the above purpose, the present invention adopts the following technical scheme. In a first aspect, the present invention provides an enhanced lysate for plasmid DNA extraction comprising 0.1-0.3mol/L sodium hydroxide, 0.5-2% Sodium Dodecyl Sulfate (SDS) and 0.1-0.5% quaternary ammonium salt. Wherein the quaternary ammonium salt is selected from one or more of benzalkonium bromide, polyhydroxy propyl dimethyl ammonium chloride, dimethyl diallyl ammonium chloride, bisdodecyl dimethyl ammonium chloride and octadecyl dimethyl benzyl ammonium chloride. Further, the enhanced lysate further comprises 0.1-1mol/L sodium succinate. According to a specific embodiment of the invention, the enhanced lysate comprises 0.2mol/L sodium hydroxide, 1% sodium dodecyl sulfate, 0.5mol/L sodium succinate and 0.2% quaternary ammonium salt. In a second aspect, the present invention provides a method for plasmid DNA extraction comprising: (1) Adding the heavy suspension into a bacterial sample which is obtained by centrifuging and enriching 100-500ml of bacterial liquid, and re-suspending and scattering bacterial bodies; (2) Adding the enhanced lysate into the re-suspension bacteria liquid in the step (1), and gently reversing and uniformly mixing to obtain a lysate; (3) Adding the neutralization solution into the cracking product, and gently mixing until egg-shaped aggregation is formed; (4) Centrifuging, transferring the supernatant to a new centrifuge tube, adding an endotoxin-removing reagent and a proper amount of isopropanol, and uniformly mixing; (5) Adding all the liquid in the step (4) into a silicon matrix column, and centrifuging to remove waste liquid; (6) Adding rinsing liquid into the silicon matrix column, and centrifuging to remove waste liquid; (7) Emptying the centrifugal column, and discarding residual liquid; (8) And adding a proper amount of preheated eluent into the silicon matrix column, and cent