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

✍ Scribed by A. Betcke; M. Pfeifer; Ch. Pöhlmann; M. Kurth; M. Hartmann; D.-H. Liebscher

Publisher
John Wiley and Sons
Year
2007
Tongue
English
Weight
819 KB
Volume
20
Category
Article
ISSN
0233-111X

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✦ Synopsis

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.

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