I'm trying to deal with variability in my ddPCR droplet counts.

Have others found success with this method? Does the temperature hold result in more false positives? Using a bio-rad QX200 system

From Rowlands et. al 2019

•To achieve optimal accepted droplet number of approximately 20,000 droplets per well, we found that it was important to incubate ddPCR plates on the cycler at 12 °C for a minimum of 4 hours post cycling and prior to transfer to the droplet reader (Fig. 11).

•Immediately after PCR, we found there to be condensation on the well walls which we believed originated from and lead to shrinkage of the droplets. The QX200 droplet reader, apart from fluorescence intensity, measures the size and shape of each droplet as they pass the detector; droplets are excluded if they do not meet quality metrics (Bio-Rad Bulletin 6407 RevA, page 20).

•If droplets shrink in size, they can be rejected by the reader resulting in fewer accepted droplets per reaction. Leaving the post-PCR plates at 12 °C for a minimum of 4 hours allowed the condensation to be reabsorbed into the droplets and lead to significantly higher passing droplet numbers for each well in the plate with no negative effects on the fluorescent signal (Fig. 11).

•The average droplet number per well was 14913 ± 2134 for reading done immediately after PCR(Fig. 11a), and 19953 ± 864 for reading done after >4 h incubation at 12 °C(Fig. 11b).