Reagent and method for extracting plasmids

A technology for extracting plasmids and reagents, applied in the field of molecular biomedicine, can solve the problems of adding additional equipment, prolonging plasmid extraction time, increasing the complexity of plasmid extraction, etc., and achieving rapid separation and removal, short time consumption, and mild operating conditions. Effect

Active Publication Date: 2015-03-04
HANGZHOU KMB BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This step not only increases the complexity of plasmid extraction and additional equipment, but also prolongs the time of plasmid extraction

Method used

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  • Reagent and method for extracting plasmids
  • Reagent and method for extracting plasmids

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Reagent for removing endotoxin in plasmid and preparation method thereof

[0039] The reagent for removing endotoxin in the plasmid includes an endotoxin-removing particle solution including complex particles capable of specifically binding to endotoxin.

[0040] The composite particle includes a specific binder capable of specifically binding to endotoxin and a carrier for bearing the specific binder.

[0041] The specific binding agent is selected from at least one of the following: (1) a polypeptide capable of specifically binding endotoxin; 2) an antibody capable of specifically binding endotoxin.

[0042] The polypeptide sequence capable of specifically binding endotoxin is selected from at least one of the following sequences:

[0043] sequence 1 keegldhsnrkn fklnrkliif fegrrikgnk;

[0044] Sequence 2 gkpsegqpse sqpsesgdsg qsspskdeng kqpektp.

[0045] The antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies or their mix...

Embodiment 2

[0053] Embodiment 2 plasmid extraction reagent and preparation method thereof

[0054] The reagents for extracting plasmids described in this embodiment include: cell suspension; cell lysate; buffer; washing solution 1; washing solution 2; eluent; endotoxin removal particle solution; plasmid binding solution. in:

[0055] Cell suspension contains 2mmol / L KH 2 PO 4 , 10mmol / L Na 2 HPO 4 12H 2 O, 137mmol / L NaCl, 2.7mmol / L KCl, 1% Tween20, pH6.0-8.0; use to add RNase A to the above mixture, mix well, and store at 4°C. Use ultrapure water as the solvent.

[0056] The cell lysate includes 0.1mol / L NaOH, 0.05% SDS (mass volume percentage), and uses ultrapure water as a solvent. Store airtight at room temperature.

[0057] The buffer contains 0.1mol / L (NH 4 ) 2 HPO 4 ·NH 4 h 2 PO 4 , 0.5mol / L KCl, pH 3, with ultrapure water as solvent. Store airtight at room temperature.

[0058] Washing solution 1 includes 2M GuHCl, 200mmol / L Tris, 2M EDTA, 1.5mol / LNaCl, pH6.8, using...

Embodiment 3

[0063] Example 3 Plasmid extraction reagent added with endotoxin removal solution and preparation method thereof

[0064] The reagents for extracting plasmids described in this embodiment include: cell suspension; cell lysate; buffer; washing solution 1; washing solution 2; eluent; endotoxin removal particle solution; plasmid binding solution; endotoxin removal solution.

[0065] Cell suspension contains 2mmol / L KH 2 PO 4 , 10mmol / L Na 2 HPO 4 12H 2 O, 137mmol / L NaCl, 2.7mmol / L KCl, 1% Tween20, pH6.0-8.0; use to add RNase A to the above mixture, mix well, and store at 4°C. Use ultrapure water as the solvent.

[0066] The cell lysate includes 2.5mol / L NaOH, 1% SDS (mass volume percentage), and ultrapure water as a solvent. Store airtight at room temperature.

[0067] The buffer contains 0.5mol / L (NH 4 ) 2 HPO 4 ·NH 4 h 2 PO 4 , 3mol / L KCl, pH 53, with ultrapure water as solvent. Store airtight at room temperature.

[0068] Washing solution 1 includes 8M GuHCl, 20...

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Abstract

The invention provides a reagent and a method for extracting plasmids. The reagent for extracting plasmids comprises a bacteria lysis solution, an endotoxin removing grain solution, an endotoxin removing solution, a plasmid combining solution, a cleaning solution, an eluant and the like. According to the reagent and the method for extracting the plasmids provided by the invention, the operation steps are simple, the time for extracting is short, the endotoxin content in the extracted plasmids can be effectively reduced, the content of endotoxin in the extracted plasmids can be reduced to be lower than 0.1EU / mu g to satisfy the clinical criteria and the obtained plasmids have high DNA (Deoxyribonucleic Acid) yield and good purity.

Description

technical field [0001] The invention relates to plasmid extraction in the field of molecular biomedicine, in particular to a reagent and method for removing endotoxin in plasmid extraction. Background technique [0002] Plasmid DNA is the most common carrier of genetic engineering. It can send a useful target DNA fragment into recipient cells for reproduction and expression through recombinant DNA technology. Therefore, the quality of plasmid extraction plays a key role in the success of subsequent molecular biology experiments. At present, many domestic and foreign companies have developed and provided plasmid extraction kits, but when these kits extract plasmids, they need to go through cumbersome steps and take a long time. And many poisonous chemical substances are adopted in the test kit, which will pollute the environment and threaten the health of the operator. [0003] Endotoxin is a component in the cell wall of Gram-negative bacteria. It is a lipopolysaccharide, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 林源吉吕航
Owner HANGZHOU KMB BIOTECH
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