ABSTRACT
Preparation of the probe usually involves three steps: incorporation of label, removal of nonincorporated precursors and measuring the efficiency of incorporation. Size is important because the rate of hybridization depends on the length of the probe. RNA probes are usually obtained by run-off transcription of cloned DNA from vectors that carry phage promoters. This allows either sense or antisense RNA to be transcribed from the same plasmid and means that the direction of cloning of the insert is irrelevant for generating probes. The size of nucleic acid is unchanged by the labeling procedure which is useful if the labeled products are to be used as molecular weight markers, but means that if the size of the probe is to be reduced, it must be done before rather than during labeling. Labeling DNA or oligonucleotides at the 3' end depends on the ability of the enzyme terminal transferase to add nucleotides to the free 3' hydroxyl in a template-independent manner.
