Molecular Cloning of ARFGEF3 Gene Associated with Breast Cancer

This assignment details the molecular cloning of the ARFGEF3 gene, focusing on its association with secondary breast cancer in the brain. The study involves several key steps, including the preparation of LB medium with ampicillin resistance, transformation of the pFLAG vector into E. coli competent cells, and the isolation and purification of the pFLAG plasmid. The assignment also covers the digestion of pFLAG vectors using restriction enzymes, agarose gel electrophoresis, and gel extraction of samples. Primers were designed for PCR amplification of the ARFGEF3 gene, followed by touchdown PCR and cloning into the pGEM vector. The results section highlights the successful transformation of pFLAG into E. coli, plasmid isolation, and restriction digestion analysis. The validation of cDNA and mutations was performed using PCR and Sanger sequencing. The assignment aimed to validate the gene insert and evaluate mutations in comparison to the normal gene, providing insights into potential biomarkers for predicting the risk of breast cancer brain metastases. The methodology includes a detailed flowchart summarizing the cloning process and the experimental procedures used to achieve the objectives of the study.