EFFECTS OF MOLAR INSERT TO VECTOR RATIOS ON CLONING EFFICIENCY USING E. COLI TRANSFORMATION

Abstract

Cloning efficiencies, or relative successful recombination events, are a function of the molar ratios of insert to vector when used in Escherichia coli transformation. Using Ml3mp and pBR322 vectors for transformation, a range of insert fragment sizes of 123bp, 1Kb, 4Kb, and 6Kb have been studied for their relative cloning efficiencies over a range of molar insert to vector ratios of 1:1, 2:1, and 10:1. The 123bp, 1Kb, and 4Kb insert fragments were analyzed individually with the Ml3mp vector M13mp19RF and the 6Kb insert fragment was analyzed with a pBR322 vector. For each insert fragment, a phosphorylated and dephosphorylated vector was tested for DNA fragments having both "sticky-ends" (staggered double-strand cleavages in DNA) and "blunt-ends (non-staggered double-strand cleavages in DNA). T4 DNA ligase was used for circularization of recombinant DNAs. E. coli HB101 competent cells were transformed with each vector in this study for plasmid amplification. Three conclusions became apparent from this study: as insert fragment size increases, within a given insert to vector ratio, the insertion efficiency decreases; as the insert to vector ratio increases, within a given insert fragment size, the insertion efficiency remains approximately equal or increases; and treating the vector with bacterial alkaline phosphatase (BAP) decreases the recircularized vector molecule background (i.e. vector molecules without insert) in comparison to vector not treated with BAP.