Abstract
An automated apparatus, designed and constructed for use in fast time-resolved studies of mammalian DNA repair after u.v. irradiation, is described. The methodology makes use of caged DNA-break-trapping reagents, e.g. [alpha-32P]dideoxyadenosine triphosphate [see Meldrum, Shall, Trentham and Wharton (1990) Biochem J. 266, 885-890] and the apparatus incorporates excimer lasers both for the delivery of u.v. damage and for the photoactivation of the caged reagents. The design is based on a sample-changing turntable which permits the sequential irradiation and quenching of samples. A maximum of eight samples can be processed in a single experiment, the sequence of events being programmed on a microcomputer, which permits a very generalized experimental sequence. Pipettes for addition of cells, nucleotides and quenching agent are driven pneumatically. A pair of pneumatically operated platinum electrodes allows electroporation of cells for loading of negatively charged reagents prior to irradiation. The time resolution of the apparatus is dependent upon the experimental scheme used and can be very short (e.g. 1 ms) when caged reagents are used; a more usual period is 1 s for the shortest incubation.