Reassociation kinetics hydroxyapatite

Fig. 1. Reassociation kinetics of nucleic acids isolated from various sources. Incubation reactions were usually carried out in 0.12 M phosphate buffer at 60°C before analysis on hydroxyapatite. Total mouse DNA, 2 mg/ml, was mixed with H-labeled single copy DNA, 1 ju.g/ml, previously fractionated at Cat 220 and E. coli [ C]DNA, 10 /ng/ml, before reassodation to the Cat values shown. The MS-2, poly(U) - - poly(A) and T< reassociation profiles are from Britten and Kohne (1968). The AT-rich satellite DNA was isolated from total mouse DNA by CsCl gradient centrifugation before reassociation as described previously (Church, 1973). All DNA s were sheared to a single-stranded molecular weight of approximately 120,000 before being heat denatured for 10 minutes at 100°C in 0.03 M phosphate buffer.

Fig. 2. The kinetics of reassociation of calf thymus DNA measured with hydroxyapatite. The DNA was sheared to small fragments and the single-stranded fragments incubated under renaturing conditions. At various times samples were passed over a hydroxyapatite column at 60 C, under conditions in which only duplex DNA binds to hydroxyapatite. Because of the large range in Cot values, different initial concentrations (Cq) of calf DNA were used to obtain the complete renaturation curve A, 2 jug/nni , 10/xg/ml o, 600Mg/ml and 8,600 /ug/ml. The crosses are radioactively labeled E. coli DNA at 43 jug/ml present in the renaturing mixture of calf thymus DNA at 8,600 The rate of renaturation of E. coli DNA provides an internal standard with which the...

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