A Highly Efficient Method for the Construction of a Plasmid-Based cDNA Library

Abstract

We have developed a highly efficient method for the construction of a plasmid-based cDNA library. The new method was based on the addition of complementary single stranded oligomers to cDNA and vector as 5' overhangs and annealing of the 5' overhangs before ligation to increase ligation efficiency. To generate the long, complementary 5' overhangs to cDNA and vector DNA a common un phosphorylated adaptor composed of 8 bases (Ad-B) and 12 bases (Ad-T) of two complementary oligomers was ligated to the cDNA and vector. Only one of the two adaptor stands was ligated to either cDNA or vector. The annealing of the 12 bases of 5' overhangs prior to ligation greatly simplified the ligation step and increased the efficiency of ligation reaction. The ligated DNA was then transformed into E. coli by the electroporation method. By this method we have constructed two plasmid-basmid cDNA libraries of approximately 5 X 106 and 5 X 107 cfu from 100 ng cDNA made from the flower bud and leaf tissues of chinese cabbage (Brassica campestris L. ssp. pekinensis) using pBluescript KS( + ) as a cloning vector, respectively.