Construction and Analysis of A Directional Xenopus laevis Embryonic cDNA Library for Yeast Two-hybrid Using Modified Random Primers

Here we describe a new strategy for cDNA library construction, in which the random primers directing the first strand cDNA synthesis contain additional d (AC) at their 5′-ends, and the linkers which will be added to the double strand cDNA contain d(GTCG) at the 5′-ends. When the linker is added to the 3′-end of the cDNA, a complete SalI site d(GTCGAC) will form at the 3′-end but not at the 5′-end of the cDNA fragment. After SalI digestion, the 3′-cohesive and the pre-existing 5′-EcoRI cohesive ends are used to introduce the double stranded cDNA into the linearized plasmid vectors. The ligation products were used to transform E.coli to produce a cDNA library. Using this method, we constructed a directional Xenopus laevis embryonic cDNA library for yeast two-hybrid. We checked the ratio of empty vectors, the size of inserts and existence of several genes, the results showed that cDNA library construction was successful.