Abstract
Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a simple and environmentally friendly protocol for recovering DNA from ddPCR.
Highlights
SUMMARY The coalescence of the Droplet DigitalTM PCR emulsion droplets is achieved by rapid freezing in liquid nitrogen
Droplet microfluidics (DM) technology has proved to be a unique and versatile tool for a broad range of biological assays. It relies on the physicochemical properties of two immiscible liquids and their manipulation through interconnected microfluidic channels, enabling the generation and manipulation of small and separated volumes [1,2,3]
Our reference method for DNA retrieval was the chloroform protocol described in the Bio-Rad ddPCR application guide [16]
Summary
Our reference method for DNA retrieval was the chloroform protocol described in the Bio-Rad ddPCR application guide [16] This protocol consists of combining the emulsion replicates, removing excess oil and homogenizing the sample with Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and chloroform (Sigma-Aldrich, MO, USA). These steps were followed by a high-speed centrifugation to separate the phases into a lower oil phase and an upper aqueous phase, with the latter containing the DNA. Whereas Chl:O 2 was necessary for a clear phase separation
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