BATCH PROCEDURE FOR THERMAL ELUTION OF DNA FROM HYDROXYAPATITE

Thermal elution of reassociated DNA on hydroxyapatite columns is time-consuming because it is difficult to run more than two samples simultaneously. A batchwise hydroxyapatite thermal elution method that allows simultaneous handling of up to ten samples is described. In this method, DNA samples are mixed with hydroxyapatite contained in test tubes. In each elution step the test tubes are rapidly centrifuged in a heated centrifuge and wash solution is decanted. Fresh, prewarmed wash solution is added and mixed with the hydroxyapatite pellet. This centrifuge method was used successfully for: (a) fractionating single- and double-stranded DNA in reactions exhibiting low and high levels of reassociation; (b) thermal elution reactions using DNA from the same or different bacteria; (c) time-course reassociation studies; (d) reactions carried out at different incubation temperatures. Sensitivity and reproducibility of the centrifuge hydroxyapatite assay were identical to those observed in the column assay. © 1969.