Abstract

Transformation is one of the fundamental and essential molecular cloning techniques. In this paper, we have reported a modified method for preparation and transformation of competent cells. This modified method, improved from a classical protocol, has made some modifications on the concentration of calcium chloride and competent bacteria solution, rotation speed in centrifugation and centrifugation time. It was found that the optimal transformation efficiency were obtained when the concentration of CaCl₂ was 75 mmol/l, OD₆₀₀ of the culture meets 0.35 to 0.45, the temperature of rotation was 4°C , rotation speed was 1000 g and rotation time was 5 min. Even more, we also found out that the transformation efficiency would increase about 10 to 30 times when adding 15% glycerine into CaCl₂ solution. The transformation efficiency, using our new method, reaches 10⁸cf u/μg and higher than ultra-competent Escherichia coli method. This method will improve the efficiency in the molecular cloning and the construction of gene library.

Key words: Competent cells, CaCl₂, improved method, transformation, glycerine, transformation efficiency.