U324650.pdf (58.26 MB)
The construction and use of lambda vectors in molecular cloning.
thesisposted on 2015-11-19, 09:08 authored by Wilhelmine A. M. Loenen
A multipurpose vector vas constructed which can be used for cloning DNA fragments of about 20 kb generated by restriction enzymes EcoRI, HindIII, BamIII and enzymes producing the same sticky ends (e.r. BgIII, Bell. MboI and Sau3A). Recombinants can be selected by their Spi- phenotype and their pronagation is facilitated by the presence of a chi-site. This choice of enzymes simplifies the cloning procedure considerably and makes this vector particularly suitable for the establishment of gene banks. It can be used in simple cloning experiments, selecting recombinants on a (P2)lysogen as well as for highly efficient cloning after removal of the vector's central fragment and size selection of the donor DNA. This vector can also be used as an expression vector when cloning into the vector's sites using the powerful ? promoter for leftward transcription, PL and the phage's antiterminator function, N. Recombination-deficient hosts for the propagation of recombinants have been investigated to minimize undesired genetic rearrangements. The absence of restriction targets for several enzymes in the vector arms led to attempts to clone the genes for these enzymes and their modification enzymes. When these failed, the genes of Providencia stuartii l64 were used as a model system to study the behaviour of such genes when carried on a ? vector. These genes turn out to be highly unstable in such a situation and experiments suggest a possible negative influence of the modification activity on host and viral functions.
Date of award1982-01-01
Awarding institutionUniversity of Leicester