Authors: IRFAN AHMAD, TEHSEEN RUBBAB, FARAH DEEBA, SYED MUHAMMAD SAQLAN NAQVI
Abstract: Bacterial transformation is a significant breakthrough in the field of molecular biology for cloning purposes. A variety of chemical and physical methods are utilized for enhancing bacterial competence. The present study was designed, via the consolidation of chemical and physical methods, to improve competence of cells for high transformation efficiency up to 10^9 transformants/µg of DNA. LB and SOC media and their supplemented combinations with osmotic agents (sucrose and glycine) were used to prepare electrocompetent cells of E. coli DH5\alpha at temperatures of 25 and 37 °C, which were then subjected to electroporation. The results showed that cells grown in SOC medium showed high growth rates and increased transformation efficiencies as compared to LB, while media supplemented with osmotic agents also showed higher transformation proficiency as compared to the controls. In all combinations tested, the highest transformation efficiencies were observed in the cells grown in SOC-SG at 25 °C, i.e. 3.56 × 10^9 cfu/µg of DNA. This study provides valuable knowledge for low-budget biotechnology labs to prepare competent cells with efficiency comparable to commercially available competent cells.
Keywords: Electroporation, optimization, culture conditions
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