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When should I choose 5’ or 3’ labeling?

It makes no difference to your assay sensitivity or protocol whether you choose 5' or 3' labeling. Both 5' and 3'-labeled probes give you the option of labeling the probe enzymatically with another label at the opposite end of the probe. However, 5'-labeled probes are easier to synthesize than 3'-labeled probes and are therefore slightly cheaper. We generally recommend using 5'-labeled probes unless there is a good reason to do otherwise.