Bacterial, Phage and Molecular Genetics: An Experimental by Professor Dr. Ulrich Winkler, Doz. Dr. Wolfgang Rüger, PDF

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0. uncorr. 0. 0. ; DNA) will be plotted graphically as a function of the wave length. 36 5. Titering. Dilute the dialyzed phage suspension 10- 8 and 10- 9 in P-buffer. 2 ml of E. coli BA as an indicator by the soft agar overlay technique on nutrient agar. Dilution Plate No. 10- 8 1 10- 8 2 10- 9 3 10- 9 4 Plaques Titer After 18-hrs incubation at 37 0 C plaques will be counted and the titers calculated. Evaluation 1. Degree of concentration and phage yield. Compare titer A of the dialyzed phage suspension with titer B of the mixture of crude lysates: A B = Compare Volume C of the mixture of crude lysates used with Volume D of the dialyzed phage suspension: ml ml If the ratios differ, discuss possible reasons.

Evaluation. The next day count the plaques on plates No. 1-4 and calculate the phage titer of the crude lysate. 69 For further details see Appendix. Preparation of high titer stocks of temperate phages requires induction of the culture lysogenic for this phage by UV-irr~dia­ tion or, if the prophage has a temperature-sensitive repressor, a short heat treatment followed by incubation until lysis. Another technique is the following: Two drops of an overnight culture of a phage-sensitive bacterial strain and 10 5 to 10 6 phage particles are mixed in 3 ml of NB soft agar and are layered onto a NB agar plate.

A intracell. X174, fd extracell. intracell. extracellular intracellular DNA isolated from phage particles DNA isolated from infected bacteria 3. The sedimentation constant and the density of macromolecules can be determined by u1tracentrifugation. In an analytical ultracentrifuge the distribution of the molecules in the centrifugation tube can be observed optically befOre, during and after centrifugation. Which two types of centrifugation are represented in the graphs below and how are they characterized.

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