Colonies were picked in up to 10 μl of media and added to 20 μl of QuickExtract Solution (Lucigen, USA) to extract DNA. DNA was diluted 1:10 before PCR. JAK2V617F (SNP ID: rs77375493) mutant and wild-type specific fluorescent probes were obtained from Thermo Fisher Scientific (#4351379). For droplet generation and analysis, we used the QX200 Droplet Digital PCR Systems consisting of two instruments: the droplet generator and the droplet reader. The droplet generator divided the sample by creating 20,000 partitions (droplets). The droplets were then transferred into PCR plates and, at the end of the amplification cycles, placed into the droplet reader, where each droplet is read as mutated or wild-type by issuing specific fluorescence signals (FAM for the mutation and Hex for the wild type). These signals, after being counted, were redistributed according to the Poisson’s law. All reagents were purchased from Bio-Rad. Quantitative PCR was performed with annealing/extension temperatures of 59°C × 40 cycles. The VAF was determined from the fraction of the single-allele droplets containing the variant allele.

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