Cholate and pH reduce interference by sodium dodecyl sulfate in the determination of DNA with Hoechst.
The use of the fluorescent dye 33258 Hoechst (Hoe) to quantitatively determine DNA in cell culture in the presence of lysing agents like sodium dodecyl sulfate (SDS) is limited by the masking effect of high levels of nonspecific fluorescence, caused by the binding of Hoe to micelles. The masking effect can be reduced substantially by increasing the concentration of the counterion, the addition of cholate, or the pH of the buffer. An optimized method was developed, combining the antimasking effects of sodium chloride, cholate, and pH to accurately determine DNA concentrations as low as 15 ng/ml in the presence of up to 6.9 mM (0.2%) SDS. The effectiveness of SDS in cell dissolution can now be combined with the specificity and sensitivity of Hoe to determine cellular DNA.
Bester
M J
MJ
Department of Chemical Pathology, University of Pretoria, South Africa.
Potgieter
H C
HC
Vermaak
W J
WJ
D000818:Animals; D006690:Bisbenzimidazole; D002417:Cattle; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D019826:Cholic Acid; D002793:Cholic Acids; D004247:DNA / Q000032:analysis* / Q000592:standards; D004273:DNA, Neoplasm / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008722:Methods; D008823:Micelles; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured / Q000737:chemistry
D002593:Cetrimonium Compounds; D002793:Cholic Acids; D004273:DNA, Neoplasm; D008823:Micelles; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D019826:Cholic Acid; D006690:Bisbenzimidazole; D000077286:Cetrimonium