The gel imaging system can achieve accurate molecular weight quantification and concentration analysis through the combination of optical imaging and software analysis. Its core process and technical points are as follows:
1、 Principle and steps of precise quantification of molecular weight
Construction of standard curve
The system uses DNAMarker bands with known molecular weights as references, annotates their positions through software, and generates a fitting curve of molecular weight migration distance. This curve is based on the linear relationship between molecular weight and mobility in electrophoresis, ensuring the accuracy of unknown band molecular weight calculations. For example, after the molecular weights of marker bands (such as 100bp, 500bp, 1000bp) on DNA film are labeled, the system automatically fits the curve for subsequent molecular weight estimation of unknown bands.
Calculation of unknown band molecular weight
Place the gel of the sample to be tested after electrophoresis in the imaging system, and excite fluorescence through ultraviolet or visible light source (such as EB staining). After the software captures the image, calculate its molecular weight according to the migration position of the unknown strip in the gel and the standard curve. This method is more accurate than naked eye estimation, and the error can be controlled within ± 5%.
2、 Accurate quantification method for concentration
Density calibration and comparison
The system first calibrates a standard band with a known concentration (such as the DNA content of a certain band on a DNA film), and then clicks on the unknown band through software to compare its density difference with the standard band and calculate the concentration of the unknown sample. For example, if the standard band density is 100 units, corresponding to a concentration of 50ng/μ L, and the unknown band density is 50 units, then its concentration is approximately 25ng/μ L.
Linear relationship of optical density
The absorption of light by the sample is linearly related to concentration or mass. The system calculates the unknown sample concentration by measuring the optical density (grayscale value or fluorescence intensity) of the strip, combined with standard curves or known sample data. This method is applicable to the concentration determination of DNA, RNA and protein gel (such as PAGE gel).
3、 Key technical support
Optical system optimization
Using ultraviolet or white light transmission sources to ensure uniform light distribution and reduce the impact of uneven light density on the results. High resolution CCD or digital camera captures images, combined with dark box design to shield external light source interference and improve imaging quality.
Software analysis function
The software supports operations such as background subtraction, strip segmentation, and density scanning, and can generate longitudinal scan curves and integrate them to achieve accurate quantification. For example, in PCR quantification, software can calculate the relative percentage of each band to the total band, while density scanning generates a longitudinal distribution curve of band grayscale values.
4、 Application scenarios and advantages
Molecular biology research: precise determination of DNA/RNA molecular weight and concentration, assisting in gene cloning and sequencing validation.
Protein engineering: Quantitative analysis of the proportion of protein expression products and optimization of fermentation processes.
Drug development: Evaluate drug purity through concentration determination to ensure quality control.
This system achieves dual precise quantification of molecular weight and concentration through standardized processes and advanced technology, providing reliable data support for life science research.