相关实验视频
Updated: May 22, 2025

07:10
Agarose Gel Electrophoresis for the Separation of DNA Fragments
Published on: April 20, 2012
763.0K
光染料分子重量对用毛细血管电泳分离DNA的作用
Yuqing Xu1, Xin Chen2, Shuaiqiang Fan1
1Engineering Research Center of Optical Instrument and System, Shanghai Key Lab of Modern Optical System, Ministry of Education, University of Shanghai for Science and Technology, No. 516 JunGong Road, Shanghai, 200093, China.
Journal of fluorescence
|March 13, 2025
概括
本研究将光染料与双链DNA (dsDNA) 的毛细电泳 (CE) 进行比较. 由于其较低的分子量,EvaGreen提供了更好的分离,使得精确的dsDNA碎片分析.
科学领域:
- 分析化学 分析化学
- 分子生物学分子生物学
背景情况:
- 毛细管电泳 (CE) 对于双链DNA (dsDNA) 质量控制至关重要.
- 光染料在CE中用于dsDNA分离,但它们的分子重量可能会影响分离性能.
- 了解染料特异性影响对于优化dDNA分析至关重要.
研究的目的:
- 使用CE系统地比较dSDNA分离的光染料.
- 为了评估染料度和分子量对光强度和迁移时间的影响.
- 为了确定最佳的染料度,以提高dSDNA片段分辨率.
主要方法:
- 用光染料 (SYBR Green I,凝绿,EvaGreen) 进行dSDNA通过CE. Green分离的系统比较.
- 在不同染料度下分析光强度和迁移时间.
- 优化染料度用于dSDNA碎片分析.
- 使用CE. 高分辨率分离fX174-Hinc II消化剂.
- OpenCV的应用用于从电光电图中虚拟dsDNA频段的生成.
主要成果:
- 染料度在CE中显著影响dSDNA迁移时间,SYBR Green I显示出比Gel Green和EvaGreen更大的影响.
- EvaGreen的较低分子量可能有助于其分离效率.
- 确定了最佳度:SYBR绿I和凝绿的1×,EvaGreen的0.005×.
- 在dsDNA度 (0.1-0.5 ng/μL) 和光强度与SYBR Green I或Gel Green之间存在线性关系.
- 在12分钟内,只有6bp的差异就能成功地分辨出dDNA片段.
结论:
- 染料的选择和度是优化CE中dsDNA分离的关键参数.
- 由于其分子特征,EvaGreen证明了改善dSDNA碎片分析的潜力.
- 该研究提供了一个框架,用于选择适当的光染料,用于使用CE的高分辨率dSDNA分析.
相关概念视频
DNA Agarose Gel Electrophoresis
95.5K
Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify DNA fragments using a gel extraction protocol.
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA...
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA...
95.5K
Electrophoresis: Overview
833
Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There...
There...
833
Capillary Electrophoresis: Applications
312
Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
312
Capillary Electrophoresis: Instrumentation
164
Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary for the separation by connecting to an anode (the positively charged electrode) and a cathode (the negatively charged electrode) located in buffer reservoirs at each end of the capillary tube. The system includes a sample vial, a fused silica capillary tube coated with polyimide for mechanical strength through which the sample components...
164
SDS-PAGE
27.2K
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...
27.2K
Two-dimensional Gel Electrophoresis
5.7K
Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such...
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such...
5.7K

