Authors: Ali Osman BELDÜZ, Zihni DEMİRBAĞ, Sabriye DÜLGER, Hülya COŞKUNÇELEBİ
Abstract: A strategy to optimize the efficiency of the transformation of Esherichia coli JM 1 09 cells made competent using the calcium-chloride method was developed. 8571 bp M 13crp RF DNA, derived from M13mp18, was used as a vector in the transformation experiments. The cells were treated with 1 00 mM calcium chloride at different pH values. The transformation efficiency of the competent cells was improved by decreasing the pH of the CaCl_2 solution and increasing the formation of plaques per LIg vector . Moreover , after 24 h storage at +4°C, competent cells prepared with CO_2-saturated CaCl_2 had a 50% greater transformation efficiency compared with cells prepared using non-saturated CaCl_2 although, in absolute terms, this storage decreased the transformation efficiency overall. Thus, the use of CO_2-saturated CaCl_2 at pH 4.6 generally increases the transformation efficiency of E. coli cells by a factor of up to 6.3.
Keywords: Plasmid, M13, Transformation, Calcium chloride, Escherichia coli