For some time now our lab has used the GE Healthcare ECL Random-Prime Labelling and Detection System to label probes for Southern Blots instead of using radioactive label. Overall, we have been quite happy with the results that we’ve obtained using this kit. It is extremely simple to use and gives clean, strong signal. Moreover, labeling the probe with this system, which basically consists of mixing two reagents in the kit with the probe fragment, takes only 15 minutes.
The ECL Random-Prime Kit uses a fluorescein-11-dUTP that is incorporated into the DNA probe by the Klenow fragment of DNA Polymerase I. Probe that is bound to the membrane is detected using an HRP conjugated anti-fluorescein antibody. Just as in the ECL kits for Western Blots, the HRP catalyzes the breakdown of luminol, included in the kit, which produces a chemiluminescent signal that can be detected on film. Signal can be detected anywhere from 1 minute to a few hours after the blot has been incubated with substrate. Although the blot can be left on film overnight, the signal does usually fade with time – in most cases you get your results the same day.
In my experience, I have found this kit fast and easy to use. The level of signal that I get is comparable to the levels I used to get with 32P, but not having to worry about radioactive contamination or waste is a big plus. Also, according to the manufacturer, once the probes are generated, they are stable for up to 9 months (although I have not tested this yet). The one downside to this system is that it is more difficult to adjust the stringency of the hybridization.
In summary, I would say that the GE Healthcare Random-Prime Labelling Kit is quick, sensitive and easy to use. Plus, not having to worry about radioactive contamination or waste makes it much less stressful to use too.
Heran Darwin, Ph.D.
Postdoctoral Fellow
Washington University School of Medicine
St. Louis, Mo.