Methods in Microbiology, Vol. 18 by T. Bergan (Ed.)

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By T. Bergan (Ed.)

Proven virtually 30 years in the past, Methods in Microbiology is the main prestigious sequence dedicated to ideas and method within the box. Now absolutely made over, revitalized, with a brand new structure and elevated scope, Methods in Microbiology will proceed to supply you with attempted and verified, state-of-the-art protocols to without delay gain your learn.

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1. The acid denatures and depurinates the DNA so that it is in a state similar to the four deoxyribonucleotides. The concentration of the N-bromoacetamide must be such that the bromium ions are in excess of the bases. Therefore, the bromination conditions must be rechecked if the DNA concentrations are altered. N-Bromoacetamide has a slight absorbancy in the 260- to 280-nm range and, since some of it is used up in the sample cuvette, adding the lesser amount to the blank cuvette compensates for this.

1983). J . Liq. Chromutogr. 6 , 16731691. , Holder-Franklin, M. , and Franklin, M. (1977). Anal. Biochem. 81, 461-466. Chargaff, E. (1955). In “The Nucleic Acids” ( E . Chargaffand J . N. ), Vol. I , pp. 307-371. Academic Press, New York. De Ley, J. (1970). J . Bacteriol. 101, 738-754. Dunn, D. , and Hall, R. H . (1975). In “Handbook of Biochemistry and Molecular Biology” (G. D. ), Vol. 1, pp. 65-215. CRC Press, Boca Raton, Florida. , Midgett, R. , Kuo, K . , McCune, R. A , , and Gehrke, C. W.

1. Using a protein- and RNA-free reference DNA sample of known mol% G + C content (two to four of varying G + C content would be even better), calculate the absorbance ratios at the following wavelengths (nm): 2401275, 2401280, and 2451270. 28 JOHN L. 425 1. 24 From Table I1 Ulitzur (1972) + 2. Plot the values obtained in step 1 on a graph (ratios vs mol% G C) and draw lines through each of them using the pertinent slopes from Table 11. This step will normalise different spectrophotometers, which may differ in absolute values but the slopes will be the same.

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