10.2.8.2. Exonuclease-I digestion of tagged primer
Exonuclease-I specifically digests only single-stranded DNA, in a 3’-5’ direction, and thus inactivates unincorporated tagged-cDNA primer prior to PCR. This reduces ten-fold the chance of falsely detecting the incorrect strand (Craggs et al., 2001) and is a common step in strand-specific RT-PCR (Purcell et al., 2006; Lin et al., 2009; Plaskon et al., 2009; Runckel et al., 2011).
1. Add to the cDNA
10 u Exonuclease-I.
2. Incubate 30 min at 37oC; inactivate 15 min at 70oC.
3. Dilute cDNA reaction ten-fold, to 200 µl.