Directionality bias in T/A cloning

T/A cloning is a popular method for generating recombinant DNA plasmids. This method relies on single A:T nucleotide base pairs between PCR product ends and vector. Theoretically, the directionality of insert ligation with relation to the vector is random. However, we have continuously observed directionality bias using the pGEM-T Vector System for T/A cloning in a Course-based Undergraduate Research Experience (CURE). Cloning of over 400 inserts has shown directional bias higher than 74% (p-value < 0.0005) "sense" to the T7 promoter of the vector. Awareness of biased insertion in our applications reduces time and cost in cloning and downstream analyses.