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We have noticed that yields of ssDNA are not significantly affected by the presence of ampicillin and/or kanamycin in the culture medium, but it ensures that all cells harbor a plasmid and, in case of MI3KO7, are infected. Preparation of Hybridization Probes, Mutagenesis, and Nucleotide Sequence Analysis The ss plasmid DNA prepared as described above is usually sufficiently pure for the preparation of hybridization probes, mutagenesis, and sequencing? °,12 Due to the presence of ss helper phage DNA, it is advisable to perform the primer hybridization step more stringently than is needed using filamentous phage vectors.
Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," p. 90. , Cold Spring Harbor, New York, 1982. [2] PLASMIDS FOR THE PRODUCTION OF s s D N A 29 Messing in Vol. 101 of this series. ~° The unique restriction enzyme cleavage sites, present in the multiple cloning sites linker of pKUN9 and pKUN19, are shown in Fig. 2. Competent cells are prepared by inoculation of the appropriate volume of 2x YT medium with a colony of JM101 or JM101[pCU53]. Grow under aeration at 37° to a density of 5 x l07 cells/ml.
68, p. 379. ~0j. W. Szostak, J. I. -K. Tye, P. Chiu, F. Sherman, and R. Wu, this series, Vol. 68, p. 419. H D. Hanahan and M. Meselson, this series, Vol. 100, p. 333. 12 L. Clarke, R. Hitzeman, and J. Carbon, this series, Vol. 68, p. 436. ~3 D. Anderson, L. Shapiro, and A. M. Skalka, this series, Vol. 68, p. 428. ~4D. A. Kaplan, L. Greenfield, and R. J. Collier, this series, Vol. 100, p. 342. 15 L. Clarke and J. Carbon, this series, Vol. 68, p. 396. ~6 H. C. Birnboim, this series, Vol. 100, p. 243.
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