Nuclease assay for TaqMan qPCR probes

(notes from IDT Research Dept. 1/26/07)

After the probe has been ordered, an easy test that can be performed to ensure that the probe is quenching properly is to read the fluorescence from an aliquot of a probe, then perform a nuclease digestion of that aliquot and take a second fluorescence reading.

• Make two 1-mL solutions of 100nM probe in Beacon2 buffer

• To one, add 1uL (7.5U/uL) of micrococcal nuclease (or DNase). Mix well and incubate for 1hr at 37C (in the dark). Allow to cool to room temp (in the dark). Store the other tube (no enzyme) in the dark.

• Measure fluorescence/emission (495/520n) of probe with no enzyme and digested probe and calculate ratio

Beacon 2 buffer:

      10mM Tris pH 8.3, 
      50mM KCl
      5mM MgCl2
      1mM CaCl2

Sample data set of a working digestion:

 digestion 1     24427
 digestion 2     26366
 digestion 3     22415

 no digestion control   2795

ratio Digestion/Control = 8.73 +- 0.71