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By Professor Dr. Ulrich Winkler, Doz. Dr. Wolfgang Rüger, Priv.-Doz. Dr. Wilfried Wackernagel (auth.)

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Collect a total of about 35 fractions. 420 against phosphate buffer as a reference. 4, to each of the fractions 18-28. 1 ml NADH-solution. 1 ml of the diluted LDH fractions. Incubate at 37 0 • Stop the reaction at time t = 45 sec, by placing the reaction mixture into a boiling water bath for 7 min. Finally cool the samples to room temperature. Treat all 11 LDH fractions successively in this manner. As a blank use the same reaction mixture as for the LDH test but replace the LDH solution by the same amount of phosphate buffer.

3 PASTEUR pipettes. One 100-ml beaker. For several groups * In this mutant the deletions b 2 and b s , which arose independentlyfrom one another, as well as the pOint mutationc (clear plaque), were introduced into the same genome by recombination. 39 together: 1 scale. 1 refractometer with thermostat. ultracentrifuge with swinging bucket rotor SW56Ti. BECKMAN 2nd day: 1 ml of a log culture of E. coli K12s in NB medium (cell titer approx. 8 x 10 8 /ml). 5 plates with TBY-agar. 5 TBY-soft agar tubes.

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.

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