The use of radioactively labeled DNA probes is still the most sensitive method for detection of genes in Southern and Northern blots. Due to the relatively long emission times of radioactive probes in comparison to non-radioactively labeled ones, the detection of low abundant genes using long exposures as well as multiple short exposures to adjust signals of abundant genes are both possible.
The Rediprime™ II Random-Prime DNA Labeling System is available with either 30 or 60 (RPN#1633 or RPN#1634, respectively) tubes, each tube contains a single pre-mixed freeze-dried reaction. The tubes can be stored at room temperature for 6 months. The reaction mix contains a buffered solution of dATP, dGTP, dTTP, a dye, exonuclease-free Klenow fragment and random primers. Therefore, only the DNA probe and the radioactively labeled dCTP have to be added. For control labeling reactions, freeze-dried lambda HindIII control DNA (300 ng) is also provided in the kit. The company claims that due to the use of an improved exonuclease-free Klenow fragment, a high specific activity for most labeled DNAs can be expected. When using the recommended Revidue [32P] or [33P] dCTP (specific activity of 3000 Ci/mmol), labeling is completed after a 10 min incubation at 37°C, a much shorter time than protocols using conventional labeling methods. The individually-packaged, lyophilized system is optimized to minimize pipetting small volumes of reagents as well as time handling radioactivity. Therefore, possibilities for spills and spreading radioactivity when opening tubes are limited.
When setting up the labeling reaction, it is essential that the concentration of DNA probe does not exceed 25 ng in a volume of 45 µl TE buffer (10 mM Tris/HCl pH 8.0, 1 mM EDTA). Higher amounts of DNA and higher volumes, e.g. when using more dilute DNA samples, results in different reaction conditions and reduced the labeling efficiency of the DNA probe. In addition, the boiling of the DNA probe for 5 min in a waterbath is essential to allow complete denaturation; denaturation of DNA in a heat block for 5 min at 95°C also resulted in a reduced labeling efficiency of my DNA template. After denaturation, the DNA is chilled on ice for 5 min, briefly centrifuged (to collect in the bottom of the tube), and added to the reaction mix. It is important to add the radioactive dCTP (5µl) quickly and immediately mix it thoroughly with the DNA probe/reaction mix by pipetting. Since complete mixing is a critical step for optimal reaction conditions, it is best to use the recommended Revidue [32P] dCTP, which allows you to monitor complete mixing: the red solution of [32P] dCTP turns purple, when completely mixed with the Rediprime reaction/DNA solution. Incubation for at least 10 min at 37°C is recommended, but extensions of 10-30 min at room temperature might be good to test for higher incorporation rates. For my probe, I used an additional 10 min at 37°C plus 10-20 min at room temperature, before stopping the reaction with 5 µl of a 0.2 M EDTA solution. The labeled DNA probe can be either used directly for hybridization or the unincorporated nucleotides can be removed to reduce background signals on, for example, the Southern blot which is hybridized with this probe. Since my incorporation rate was mostly around 30-45%, I used the MicroSpin S-200 HR columns (GE Healthcare) to remove unincorporated nucleotides before hybridization.
For labeling of DNA probes with radioactivity, I recommend the Rediprime™ II Random Prime DNA Labeling System, since it works well, is easy to use and limits the amount of pipetting and time handling of radioactivity, thereby minimizing the risk of radioactive spills and exposure.