Factors which influence CaCl2 dependent transfection of Lambda DNA inEscherichia coli K12 recipients

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

doi:10.1002/jobm.3630200406

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1980

1981

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In vivo andin vitro recombination of Lambda DNA in CaCl2 transfection

Betcke¹,

Pfeifer²,

Pöhlmann³

et al. 1980

Z Allg Mikrobiol

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Using 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 efficiencies are 20--30% in the in vitro and in vivo recombination systems. At 30 to 37 degree C T4 ligase mainly joins natural cohesive alpha ends, while at 12 degrees C the EcoRI-generated termini are preferentially ligated to form biologically active molecules, if the cloning vector alpha 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 postinfection-dependent 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 alpha DNA fragments and the helper phage DNA.

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