The Ceriotti method (1,2) for estimating the DNA content of acid hydrolyzates (3,4) was recently improved (5) by increasing the sensitivity and range of the reaction. The influence of the chloroform extraction on the specificity of the method was defined and the probable chromogen was identified as 2-deoxyribose 5-monophosphate (5). In this paper, we use a model system for determining the primary factors influencing the recovery of DNA chromogen from protein precipitates and its subsequent reaction with indole. The goal has been to establish limits beyond which further increases in extraction time or temperature would be impractical.
MATERIALS AND METHODS
Reagents
Calf thymus DNA (Sigma) prepared by the method of Zamenhof (6) had a manufacturer's analysis: moisture (20.8%) ; total P (6.8%)l; N (13.2%) ; Na (3.75%) ; protein (0.6%) ; and N:P ratio 1.94.
Indole, crystalline (Sigma). Chloroform, Spectranalyzed (Fisher Scientific). TCA 30% (Fisher Scientific). Deoxyguanosine 5-monophosphate (d-G-5-MP), 2-deoxyribose (2-d-R) , 2-deoxy-n-ribose 5-monophosphate (2-d-R-5-MP) , and /3-lactoglobulin (P-LG) were obtained from Calbiochem. Bovine serum albumin (BSA) and casein (CAS) were Pentex and General Biochemical products, respectively.
'If the average phosphorus content of pure DNA is considered to be 9.35% then our DNA is at best only 72.7% pure. For the sake of simplicity, however, the standards have not been corrected on the basis of their P content.