User manual

Droplet Digital
™
PCR Applications Guide | 85
Fig. 9.6. Quadruplicate replicates drawn from the same poorly mixed reaction solution demonstrate
inconsistent concentration readings.
Fig. 9.7. Concentration plot of a temperature-sensitive assay (n) with a temperature-insensitive
assay (n) concentration across the plate, despite having the same amount of input DNA/well.
All sample wells were loaded with 0.5 copies/droplet Raji DNA.
Channel 1 concentration, copies/µl
0
1,20 0
Replicate 1 Replicate 2 Replicate 3 Replicate 4
Staphylococcus aureus
400
800
1,600
2,000
1,360
1,120 1,120
1,260
Effects of Poor Cycler Uniformity
If the reaction mixtures used to create technical replicates are thoroughly mixed but there
is wide variation in concentration estimates, consider the uniformity performance of your
thermal cycler. Generally, this effect is observed only when a temperature-sensitive assay
is used on a thermal cycler with poor uniformity. Uniformity at both the denaturation and
annealing/extension temperatures is important. Bio-Rad’s C1000 Touch
™
thermal cycler
with 96–deep well reaction module has excellent thermal uniformity. To test the module’s
uniformity, use the temperature-sensitive assay that has concentration variability and
create droplets from the same reaction mixture for the entire plate. Check the entire plate
for a discrepancy in concentration that exceeds the 95% confidence bounds for the wells.
If one of the block’s Peltier devices is broken or underperforming, a drop in concentration
will be consistently observed in the same quadrant(s) of the block (Figure 9.7).
Concentration, copies/µl
0
500
1 2 3 4 5 6 7 8 9 10 11 12
Sample
100
300
600
700
414
400
200
491
528
556
538
547
548
540
536
477
459
387
631
624
604
630
624
621
622
636
640
606
602
590
Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting