User manual

Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting
80 | Droplet Digital
™
PCR Applications Guide
To prevent the off-target amplification, try increasing the annealing temperature of the PCR
reaction to improve specificity (Figure 9.3), or alternatively, try digesting the sample with a
restriction enzyme that will cut the nonspecific target while preserving the specific target.
This latter approach requires some knowledge of the nonspecific target’s sequence.
Assay redesign can often be used to remove subclusters. Consider using Primer-BLAST to
determine whether one of the primers is potentially nonspecific. If this is the case, examine
whether a primer redesign would avoid the amplification of this product. If no flexibility exists
in primer design, shift the probe to minimize complementarity to the off-target sequence.
Fig. 9.2. Extra droplet clusters.
Fig. 9.3. Annealing/extension gradient where the assay detects an off-target species.
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