Molecular cloning of recombinant DNA : proceedings of the Miami winter symposia, January 1977 / edited by W.A. Scott, R. Werner ; sponsored by the Department of Biochemistry, University of Miami, School of Medicine, Miami, Florida.

Date:
1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Molecular cloning of recombinant DNA : proceedings of the Miami winter symposia, January 1977 / edited by W.A. Scott, R. Werner ; sponsored by the Department of Biochemistry, University of Miami, School of Medicine, Miami, Florida. Source: Wellcome Collection.

    121/272 page 105
    USE OF SAFER BACTERIAL HOST STRAINS 105 measured the minimal inhibitory concentrations for tetra¬ cycline and found that these were lower in cultures treated with concentrations of 50, 100 or 200 yg chloramphen- icol/ml than they were for cells incubated in the absense of chloramphenicol. TABLE 3 Survival of x2042 after 22 hours incubation in various concentrations of chloramphenicol (Cm) Concentration of Cm Viable Percent (]ig/ml) titer survival Cells grown in L broth with 100 ug DAP/ml and 4 yg Thd/ml. We next conducted a series of experiments in which x2042 cells were prelabeled .with [^Hj-thimidine, washed, and suspended in prewarmed fresh media with various concen¬ trations of chloramphenicol and [l^C]-thymidine so that we could follow the fate of [^Hj-labeled chromosomal DNA as well as [^^C] incorporation into plasmid DNA. In these experiments, we found that incubation of cells in the presence of 100 yg Cm/ml led to detectable losses in chromosomal DNA within the first several hours of in¬ cubation. Even when the concentration of chloramphenicol was reduced to 12.5 yg/ml, cell death with loss of pre¬ labeled counts became readily observable following six hours of incubation. Based on studies of these types, we found the following optimal conditions for maximal recovery of ColEl-derived plasmid vectors. A standing overnight culture is diluted 1:10 into the same prewarmed growth medium and aerated by shaking until reaching an A50O of about 0.45 ('V/S x 10^ cells/ml). Chloramphenicol is then added to 12.5 yg/ml and the culture is aerated for an
    digitised image 1
    digitised image 2
    digitised image 3