Vol. 7, Special Issue 2 (2025)

The efficacy of bacterial transformation is essential in molecular biology, especially for cloning, gene expression, and recombinant protein synthesis. This study aims to optimize heat shock parameters, that is, temperature and duration, to improve transformation efficiency in Escherichia coli DH5α utilizing the significant binary vector pBI121 (~14.7 kb), extensively employed in plant biotechnology. Competent cells were generated via a modified calcium chloride technique and exposed to nine heat shock combinations: three temperatures (37 ℃ , 42 ℃ , and 47 ℃ ) and three durations (30, 45, and 60 seconds). The success of transformation was assessed by measuring colony-forming units (CFU) per microliter. The ideal setting was determined to be 47 ℃  for 60 seconds, resulting in the highest transformation efficiency (125.6 CFU/µl), whereas the lowest efficiency (4.8 CFU/µl) was observed at 42 ℃  for 60 seconds. The findings suggest that bigger plasmids require more powerful heat shock to permeate the bacterial barrier efficiently. Although hampered by the absence of biological duplicates and plasmid quantification, this study addresses a critical methodological need for laboratories handling large plasmid vectors. It enables further procedure refinement for plant genetic engineering.