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Updated: May 22, 2025

Agarose Gel Electrophoresis for the Separation of DNA Fragments
Published on: April 20, 2012
Effect of Molecular Weight of Fluorescent Dyes on DNA Separation by Capillary Electrophoresis
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.
Abstract:
Capillary electrophoresis (CE) plays an important role in the quality control of dsDNA. So far, there has been various fluorescent dyes employed for the separation of dsDNA by CE. However, the molecular weight of the dyes may affect the mass to charge ratio of dsDNA-dye complex, consequently the separation performance of dsDNA will be changed. Herein, we systematically compared the fluorescent intensity and migration times when separating the dsDNA fragments labeled or intercalated by different dyes. Results showed that the concentration of SYBR Green I affected the migration times more than Gel Green and EvaGreen, which may be caused by the lower molecular weight of EvaGreen. The optimal concentration for SYBR Green I and Gel Green is 1×, and it is 0.005× for EvaGreen. There is linear relationship between dsDNA concentration (0.1-0.5 ng/µL) and fluorescence intensity when using SYBR Green I or Gel Green for separation. Finally, we have resolved the фX174-Hinc II digest in 0.5% HEC (1300k) containing 1× SYBR within 12 min, even though there is only 6 bp difference for the adjacent dsDNA fragments. Furthermore, we also obtained the virtual dsDNA bands by OpenCV according to the electropherogram.
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