![]() We tested the efficiency of the standard protocol with several chloroform-to-oil ratios (Chl:O), which were achieved by increasing or decreasing the chloroform volume and keeping other conditions identical. 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. Briefly, 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). Our reference method for DNA retrieval was the chloroform protocol described in the Bio-Rad ddPCR application guide. The method efficiency was calculated as the percentage of DNA recovered against the initial DNA input for each sample ( Supplementary Table 1). The initial and recovered DNA was quantified using the Qubit dsDNA high-sensitivity assay (Thermo Fisher Scientific, MA, USA). Up to three independent emulsion samples were combined into one tube to form a single replicate the number of replicates per protocol in each experiment varied from one to seven. Later, purified PCR amplicons or Qubit ® dsDNA HS Standard #2, of sizes 263 bp and 1 kb, respectively, were chosen. Initially, GeneRuler 100-bp DNA ladder (Thermo Fisher Scientific, MA, USA) was used as the DNA control, containing fragment sizes ranging from 100 to 1000 bp. To assess the efficiency of DNA recovery, DNA fragments up to 1 kb were added to the ddPCR master mix as input. The ddPCR emulsion samples were prepared with QX200 ddPCR EvaGreen Supermix and QX200 Droplet Generation Oil for EvaGreen according to the manufacturer's instructions. Iv) Transfer to silica column and proceed according to manual Iii) Homogenize by vortexing at top speed for 1 min Silica column (GeneJET PCR purification kit, Thermo Scientific) Iv) Homogenize by vortexing at top speed for 1 min Summary of methods used for breaking the ddPCR emulsion. Different chemical and physical methods were compared: chloroform, n-Octane and 1H,1H,2H,2H-perfluoro-1-octanol (PFO) as chemical treatments, and freeze-thawing and silica columns as physical methods ( Table 1). In this study we addressed the challenge of recovering DNA from droplets generated by the QX200™ ddPCR System (Bio-Rad, CA, USA). The lack of efficient methods for breaking the ddPCR emulsion forms a major challenge to using the PCR products in downstream analysis. For instance, DNA recovered from droplets has been used for production of next-generation sequencing libraries for single-cell genetics and transcriptomics studies. ![]() ![]() After the ddPCR process, the droplets are usually discarded, despite their potential as material for downstream genetic studies. However, ddPCR analysis is based on the end-point fluorescence intensity measured for each separate droplet, thereby increasing method sensitivity. As with quantitative PCR, the increase in DNA copy number due to specific amplification is revealed via fluorescent dye. ddPCR relies on DM's generation of numerous nanoliter-sized droplets, inside which independent DNA amplification reactions are carried out. The Droplet Digital™ PCR (ddPCR™) system is a droplet-based PCR platform commercialized by Bio-Rad (CA, USA) and has been widely used for pathogen detection, food quality analysis, environmental studies and medical research. Nonetheless, the most popular and well-established use of DM has been for nucleic acid detection and quantitation. Lately, DM has even been explored for studying single cells. Methods have also been developed to select and cultivate combinations of organisms for studying cell–cell interactions. The replication of microbes inside droplets has revolutionized the study of metabolism, antibiotic resistance and enzyme activity. As a result, droplets can be manipulated in a controlled manner for different ends.ĭM has been used for various purposes ranging from molecular analyses to the cultivation of individual microbes. The properties of the droplets, such as size and stability, can be adjusted by altering the microfluidic channel geometry, flow rate and reagent composition. 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. Droplet microfluidics (DM) technology has proved to be a unique and versatile tool for a broad range of biological assays.
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