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

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

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A possible explanation for this effect at the molecular level might be that excitations of the ~­ electrons of the bases (the electrons, which absorb the light) are strongly reduced in the long stacks of nucleotides in a native D~A molecule. When the optical density of a heated DNA solution is plotted against the temperature, a "melting curve" is obtained. The melting point Tm (measured in °C) of this curve is directly proportional to the GC-content and furthermore depends on the ionic strength of the DNA solution.

If the refractive index measured is higher or lower, it can be corrected by the careful addition of distilled water or crystalline CsCI. 2. 5 ml of CsCI solution containing the phages into a polyallomer tube, over layer with paraffin, tare against tubes from other groups and centrifuge in the SW56Ti rotor at 23,000 rpm (55,000 x g) for approx. 24 hrs at 20 0 C. 3. Dripping the gradient (2nd day): Pierce the tubes with the dripping device and collect fractions of 12 drops each in small test tubes.

0. 0. 0. corr. 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.

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