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[Construction of prokaryotic expression vector for MAP30 gene and study of PCR methods for rapid identification of
Dong-Hong Zhuang1, Yong-Chang Ouyang, Zhong Hu
1Department of Biology, Shantou University, Shantou, Guangdong Province 515 063, China. dhzhuang@stu.edu.cn
Abstract:
Based on the sequence reported by Lee-Huang,S, we cloned the MAP30 gene of Momordica charantia (balsam pear) into a prokaryotic expression vector pET28a (+). A method by using PCR for rapid identification of positive clone was developed. Result showed this screening method can be used to detect positive colonies from samples of bacterial, purified plasmid, liquid culture,and liquid culture treated with mixture of phenol/Chloroform. The result from liquid-culture-treated- PCR (LCT-PCR) is very close to that of by plasmid-PCR. LCT-PCR is reliable and much easier to used than plasmid-PCR, therefore the LCT-PCR can be used for clone screening during the molecular cloning.
Insights
Researchers developed a rapid PCR method for identifying positive clones during molecular cloning. This liquid-culture-treated PCR (LCT-PCR) is reliable and easier than traditional plasmid PCR for screening the Momordica charantia MAP30 gene.
Area of Science:
- Molecular Biology
- Biotechnology
- Gene Cloning
Context:
- The MAP30 gene from Momordica charantia (balsam pear) is of interest for its potential biological activities.
- Efficiently identifying positive clones is crucial for successful gene cloning and expression.
- Traditional methods like plasmid PCR can be time-consuming and labor-intensive.
Purpose:
- To develop and validate a rapid and reliable method for screening positive clones during the molecular cloning of the Momordica charantia MAP30 gene.
- To compare the efficacy of the novel PCR-based screening method with existing techniques.
Summary:
- The MAP30 gene from Momordica charantia was cloned into a pET28a (+) prokaryotic expression vector.
- A novel PCR-based screening method, termed liquid-culture-treated PCR (LCT-PCR), was developed for rapid identification of positive clones.
- LCT-PCR demonstrated high reliability and ease of use, comparable to plasmid PCR, across various sample types including bacterial colonies, purified plasmids, and liquid cultures.
Impact:
- The developed LCT-PCR method offers a significant improvement in efficiency and simplicity for clone screening in molecular cloning workflows.
- This technique can accelerate the process of gene cloning and recombinant protein expression.
- Facilitates faster research and development in fields utilizing Momordica charantia-derived compounds or genes.
