In vivo and in vitro recombination of Lambda DNA in CaCl2 transfection

Abstract

AbstractUsing CaCl2 mediated transfection with Lambda DNA fragments, in vitro joining by ligase and in vivo recombination with helper phage DNA are effective systems for generating artificial recombinants. Recombination effiicencies are 20–30% in the in vitro and in vivo recombination systems.At 30 to 37 °C T4 ligase mainly joins natural cohesive λ ends, while at 12 °C the EcoRI‐generated termini are preferentially ligated to form biologically active molecules, if the cloning vector λ 401 is used, which has only one EcoRI target. The ligation products were characterized by gel electrophoresis and CaCl2 transfection.For in vivo recombination a new CaCl2 transfection system was developed, termed postinfectiondependent CaCl2 transfection system, which is based on the infection of recipient cells with helper phages after transfection. In marker rescue experiments using this method not only single but also double recombination occurred between two independent Δ DNA fragments and the helper phage DNA.