GB/T 40170-2021 in English
VALIDPrinciple of plasmid extraction and testing
- Issued on:2021-05-21
- Implemented on:2021-12-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$272.00
《GB/T 40170-2021质粒抽提及检测通则》由TC387（全国生化检测标准化技术委员会）归口，主管部门为国家标准化管理委员会。
Introduction
Analysis of the core content of the standard
GB/T40170-2021 is my country's first special standard for plasmid operation, which systematically standardizes the technical requirements for the entire process from silica gel column to ion exchange chromatography column. The standard highlights three major innovations: for the first time, it clarifies the standardized operation process of magnetic bead method, establishes the supercoil ratio HPLC detection method, and stipulates the endotoxin limit of plasmids for cell therapy (<0.1EU/μgDNA).
Comparison of plasmid extraction methods
| Method | Principle | Yield (μg/mL bacterial solution) | Supercoil ratio | Applicable scenarios |
|---|---|---|---|---|
| Silica gel column method | High salt adsorption-low salt elution | 5-20 | 60-85% | Conventional molecular cloning |
| Magnetic bead method | Magnetic silanol specific binding | 3-15 | ≥90% | Micro sample/automation |
| Phenol-chloroform method | Organic phase separation | 10-30 | 70-80% | Restriction endonuclease sensitive strains |
Key detection technology points
Supercoil ratio detection
The standard innovatively adopts the Source15PHE hydrophobic chromatography column to separate supercoiled plasmids from open circular DNA through 1.5M ammonium sulfate gradient elution. A typical chromatogram should show a double peak feature. When calculating, please note:
- The retention time is 6.2±0.5min as the main peak (supercoiled)
- OD260/OD280 ratio <1.8 indicates protein contamination
- Transfection-grade plasmids require a supercoil ratio of ≥80%
Recommendations for the implementation of the standard
- The laboratory should establish a dual validation system of magnetic bead method and silica gel column method
- Limulus endotoxin testing must be added to cell therapy projects
- Long-term storage is recommended in a -80℃ ultra-low temperature refrigerator and repeated freezing and thawing should be avoided
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