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Updated: Dec 2, 2025
11:58
A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
8.5K
Dephosphorylation of Plasmid DNA
Cold Spring Harbor Protocols
|November 3, 2020
Summary
This protocol removes 5'-phosphate residues from plasmid DNA termini. This prevents recircularization during subsequent DNA ligation procedures, improving cloning efficiency.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Linearized plasmid DNA can recircularize during ligation.
- 5'-phosphate residues on DNA termini facilitate recircularization.
- Efficient DNA cloning requires suppression of unwanted ligation events.
Purpose of the Study:
- To present a method for removing 5 -phosphate residues from linearized plasmid DNA.
- To inhibit the recircularization of linearized plasmids during ligation.
- To enhance the efficiency of subsequent molecular cloning procedures.
Main Methods:
- A protocol for dephosphorylation of linearized plasmid DNA termini.
- Treatment of both protruding and blunt DNA termini.
- Application of the method prior to ligation reactions.
Main Results:
- Effective removal of 5 -phosphate groups from plasmid DNA.
- Significant reduction in plasmid recircularization.
- Improved yields of desired ligation products.
Conclusions:
- The presented protocol effectively removes 5 -phosphate residues.
- This method is crucial for suppressing plasmid recircularization.
- The protocol is valuable for optimizing DNA ligation and cloning workflows.
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