Primer P3 5′-CAGCAAAACCAGACGCGCG-3′ was designed from the DNA sequence determined by sequencing pHC7S with primer P2 ( Fig. 1): P1 5′-GCCACCTGACGTCTAAGAAACC-3′, and P2 5′-GCTACCCCTTCTAGCCCGAGC-3′. Primers were designed which anneal to sequences in the vector pHC79 and initiate DNA synthesis into the insert ( Fig. 2.3 DNA sequence analysisĭeletion derivatives of the small plasmid pHC7S, were constructed by restriction digestion, with EcoRI or EcoRI and NcoI (followed by filling in of the recessed 3′ termini in the case of the double digest) and ligation. typhimurium LT7 was transformed by electroporation. Each 50 µl PCR reaction contained 1 pmol of each primer and 10 or 100 ng of template DNA. The primers were those used for sequencing (see below). The samples were incubated for 90 s at 90☌, followed by 30 cycles of 90☌ for 30 s, 55☌ for 30 s and 72☌ for 30 s with a final extension period of 5 min at 72☌. PTC-100 programmable thermal cycler was used for PCR. Probes were made by random primed incorporation of DIG-dUTP and detected using a chemiluminescent detection kit (Boehringer). DNA to be probed was transferred to nylon membranes. Unless otherwise stated standard molecular techniques were used. Carbenicillin (250 µg ml −1) was used to select for the presence of the β-lactamase gene in the cosmid vector. Bacteria were cultured on L-agar or L-broth at 37☌. 2 Materials and methods 2.1 Bacterial strains, bacteriophage and culture conditionsīacterial strains and bacteriophage are listed in Table 1. Pre-emptive strategies, of general applicability, are discussed for its prevention. In a series of sequential overnight growth experiments, the basis of this selective advantage was identified. Here we report an example in which the degree to which the Salmonella library represented the genome was severely compromised by the presence of a single clone with a strong selective advantage expressed during the growth phase of the selection cycles. When selection of a desired phenotype necessitates an iterative procedure involving cycles of enrichment selection followed by growth, selection may occur in the growth phase as well as in the selection phase. We have attempted to select invasion-complemented Salmonella typhimurium recombinants by multiple passage of a library from a virulent strain through a gut organ culture system. This can be powerfully exploited in the genetic analysis of complex multi-factorial phenomena if two criteria are fulfilled: the library (comprising sufficient clones statistically to give 99% representation of the entire genome) must be truly representative, and an appropriate selection system must be available. Genomic library, Screening, Salmonella typhimurium, Cosmid 1 IntroductionĪn entire bacterial genome may be represented in as few as 500 clones using small cosmid vectors capable of maintaining large pieces of chromosomal DNA.
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