FAQ: Is nick translation the best way to make a labeled probe?

Nick translation is one way to make labeled probes. DNase I is used to nick the DNA after which DNA Polymerase I removes leading bases at the nick with the 5'→3' exonuclease and fills in with labeled bases. This procedure is good at making large, evenly labeled probes but the specific activity is not high. The NEBlot Kit (NEB# N1500) can be used for making high specific activity probes using random primers. Klenow Fragment (3'→5') exo- (NEB# M0212) can be used with the same procedure and a specific primer. T7 RNA Polymerase (NEB# M0251) and SP6 RNA Polymerase (NEB# M0207) and the HiScribe RNAi Transcription Kit (NEB# E2000) can be used to make RNA probes. Increased levels of detection in nucleic acid hybridization can be obtained due to the greater stability of RNA: DNA hybrids with respect to DNA: DNA hybrids (1).
(1) Zinn, K et al. (1983) Cell 34, 865-879.