An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Felipe A. Calil
Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7503-4078
Christopher D. Putnam
Ludwig Institute for Cancer Research, Departments of Medicine, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6145-1265
Richard D. Kolodner
Ludwig Institute for Cancer Research, Cellular and Molecular Medicine, Moores-UCSD Cancer Center and Institute of Genomic Medicine, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669
Corresponding Author
ORCiD: https://orcid.org/0000-0002-4806-8384
License:
This work is licensed under a CC BY 4.0 License. Read Full License
We have developed a simple procedure for determining if a plasmid DNA contains single strand interruptions that can be sealed by DNA ligase and thus contain single strand interruptions with 5'-phosphate and 3'-hydroxyl groups. In this procedure, plasmid DNAs with ligatable nicks were ligated by T4 DNA ligase, supercoiled by E. coli DNA gyrase and analyzed by agarose gel electrophoresis. Our results show that after sealing nicked circular DNA with DNA ligase, supercoiling by DNA gyrase produces a rapidly migrating DNA species that can easily be distinguished from nicked circular DNA by agarose gel electrophoresis.
Keywords
nicked circular plasmid, ligation, supercoiling
Figure 1
Loading...
Version History
CURRENT STATUS: Posted
Version 1
Posted 22 Sep, 2021
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Associated Publications
Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae
by Felipe A. Calil, Bin-Zhong Li, Kendall A. Torres, +4
Nature Communications (22 September, 2021)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Felipe A. Calil
Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7503-4078
Christopher D. Putnam
Ludwig Institute for Cancer Research, Departments of Medicine, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6145-1265
Richard D. Kolodner
Ludwig Institute for Cancer Research, Cellular and Molecular Medicine, Moores-UCSD Cancer Center and Institute of Genomic Medicine, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669
Corresponding Author
ORCiD: https://orcid.org/0000-0002-4806-8384
Version History
CURRENT STATUS: Posted
Version 1
Posted 22 Sep, 2021
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
We have developed a simple procedure for determining if a plasmid DNA contains single strand interruptions that can be sealed by DNA ligase and thus contain single strand interruptions with 5'-phosphate and 3'-hydroxyl groups. In this procedure, plasmid DNAs with ligatable nicks were ligated by T4 DNA ligase, supercoiled by E. coli DNA gyrase and analyzed by agarose gel electrophoresis. Our results show that after sealing nicked circular DNA with DNA ligase, supercoiling by DNA gyrase produces a rapidly migrating DNA species that can easily be distinguished from nicked circular DNA by agarose gel electrophoresis.
Figure 1
Loading...
Associated Publications
Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae
by Felipe A. Calil, Bin-Zhong Li, Kendall A. Torres, +4
Nature Communications (22 September, 2021)