Determining if DNA stained with a cyanine dye can be digested with restriction enzymes

April Maschmann, Cody Masters, Melissa Davison, Joshua Lallman, Drew Thompson, Kristy L. Kounovsky-Shafer

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and sensitivity for DNA. In order to determine if the DNA molecules are full length after the completion of the experiment, a method is required to determine if the stained molecules are full length by digesting DNA with restriction enzymes. However, stained DNA may inhibit the enzymes, so a method is needed to determine what enzymes one could use for fluorochrome stained DNA. In this method, DNA is stained with a cyanine dye overnight to allow the dye and DNA to equilibrate. Next, stained DNA is digested with a restriction enzyme, loaded into a gel and electrophoresed. The experimental DNA digest bands are compared to an in silico digest to determine the restriction enzyme activity. If there is the same number of bands as expected, then the reaction is complete. More bands than expected indicate partial digestion and less bands indicate incomplete digestion. The advantage of this method is its simplicity and it uses equipment that a scientist would need for a restriction enzyme assay and gel electrophoresis. A limitation of this method is that the enzymes available to most scientists are commercially available enzymes; however, any restriction enzymes could be used.

Original languageEnglish (US)
Article numbere57141
JournalJournal of Visualized Experiments
Issue number132
StatePublished - Feb 2 2018


  • BOBO-1
  • BOBO-3
  • Biochemistry
  • Gel electrophoresis
  • Issue 132
  • POPO-1
  • POPO-3
  • Restriction enzyme digestion
  • TOTO-1
  • TOTO-3
  • YOYO-1
  • YOYO-3

ASJC Scopus subject areas

  • General Neuroscience
  • General Chemical Engineering
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology


Dive into the research topics of 'Determining if DNA stained with a cyanine dye can be digested with restriction enzymes'. Together they form a unique fingerprint.

Cite this