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A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost

Abstract

Key message

In-house production of a positive selection cloning vector could be simple, efficient and low cost.

Abstract

DNA cloning technology requires a vector to harbour a gene of interest for multiplication of the gene in bacterial cells. Positive selection vector has become a popular type of cloning vector due to the simplicity and efficiency of the positive selection system. Due to the presence of a toxic gene, propagation of a commercial positive selection vector in common laboratory E. coli strains is infeasible. This study demonstrated a strategy for propagation and in-house production of a commercial positive selection vector, i.e., pJET1.2/blunt cloning vector, at low cost. This was done by insertion of a specially designed DNA fragment (MCS fragment), which can be easily removed later by EcoRV digestion, into the pJET1.2/blunt cloning vector to allow the propagation of the modified plasmid (termed pJET1.2M) in common E. coli strains. Removal of the MCS fragment from the pJET1.2M plasmid produces the pJET1.2/blunt cloning vector ready for gene cloning. The self-made pJET1.2/blunt cloning vector exhibited a cloning efficiency of 100%.

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Acknowledgements

The authors would like to thank the lab staff, especially Natasha Afzan Tamby Husin, Dahyudeen Dahlan and Muhd Shazreen Sobri.

Funding

This study was supported by Fundamental Research Grant Scheme (Reference Code: FRGS/1/2019/STG05/UITM/02/1) from the Malaysian Ministry of Higher Education (MOHE).

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Authors and Affiliations

Authors

Contributions

MPA and CYLY conceived and designed the research. ON performed the study. CYLY and MPA prepared the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Mohd Puad Abdullah or Chong Yu Lok Yusuf.

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Conflict of interest

The authors declare that they have no conflict of interest.

Data availability

Online Resource 1-3 are provided as electronic supplementary material.

Ethical approval

This study does not involve any animal or human participants.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 9728 KB)

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Nawawi, O., Abdullah, M.P. & Yusuf, C.Y.L. A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost. 3 Biotech 12, 216 (2022). https://doi.org/10.1007/s13205-022-03289-x

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  • DOI: https://doi.org/10.1007/s13205-022-03289-x

Keywords

  • DNA cloning
  • Positive selection
  • Low cost
  • pJET1.2/blunt cloning vector