Factors which influence CaCl<sub>2</sub> dependent transfection of Lambda DNA in <i>Escherichia coli</i> K12 recipients

Pfeifer, Marco; Pöhlmann, Ch.; Betcke, A.; Kurth, Marion; Liebscher, D.-H.

doi:10.1002/jobm.19800200406

Cited by 2 publications

References 16 publications

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

Betcke¹,

Pfeifer²,

Pöhlmann³

et al. 1980

J of Basic Microbiology

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Using CaCl, 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 efficiencies are 20-30% in the i n vitro and i n vivo recombination systems.At 30 t o 37 "C T4 ligase mainly joins natural cohesive 1 ends, while at 12 "C the EcoRI-generated termini are preferentially ligated to form biologically active molecules, if the cloning vector 1401 is used, which has only one EcoRI target. The ligation products were characterized by gel electrophoresis and CaCI, transfection.For i n vivo recombination a new CaCl, transfection system was developed, termed postinfectiondependent CaCI, transfection system, which is based on the infection of recipient cells with helper phages after transfection. I n marker rescue experiments using this method not only single but also double recombination occurred between two independent 1 DNA fragments and the helper phage DNA.A direct and facile method for in vitro production of hybrid DNA molecules is ligase joining that of cohesive termini generated by restriction endonuclease digestion. It is known these artificial recombinants can be propagated using a vector DNA molecule capable of replication in a suitable host cell. The DNA of bacteriophage Lambda has become a useful vector for cloning experiments since constructed derivatives of 1 DNA with only one EcoRI restriction target per chromosome have been available (MURRAY and MURRAY 1974, MURRAY et al. 1977).The experiments described here were carried out in order t o determine T, ligase reaction conditions under which the majority of the reaction products are biologically active linear 1 DNA molecules. We investigated different T, ligase reaction conditions to analyse the influence of temperature, DNA concentration and incubation time on the character of ligation products of A wild type DNA and of the cloning vector A 401. We used agarose gel electrophoresis of ligated A wild type and CaCl, transfection of 1 401 DNA fragments to characterize the joining products.Furthermore a method for generating artificial recombinants in vivo was developed: the postinfection-dependent CaC1, transfeetion system, a modification of the standard CaCl, transfection system of MANDEL and HIGA (1970), based on the infection of recipient cells with heteroimmune helper phages after transfection. In this system recombination of A DNA fragments with the helper phage DNA takes place in the recipient cells.

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