POL Scientific / JBM / Volume 10 / Issue 1 / DOI: 10.14440/jbm.2023.411
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PROTOCOLS

A strategy to recover a poor-quality ligase product

Sonia Del Prete1 Marta Gogliettino1 Gianna Palmieri1 Ennio Cocca1
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1 Institute of Biosciences and Bioresources, CNR, Naples, Italy
JBM 2023 , 10(1), 1;
Published: 16 November 2023
© 2023 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation.
We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5’ end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.

Keywords
T4 DNA ligase
Polymerase Chain Reaction (PCR)
cloning
recombinant DNA
educational guide
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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TAB, Published by POL Scientific