Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations
Published: April 30, 2023
Abstract
Source: Bonner, E. R. et al. Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids. J. Vis. Exp. (2019)
In this video, we present a step-by-step procedure to generate emulsified nano-droplets to detect target nucleic acids by digital polymerase chain reaction or dPCR.
Protocol
1. Detection and Quantification of Target DNA Using dPCR Prepare the dPCR reaction mixture for 8 wells of the PCR strip tube plus 10% extra, by adding 220 µL of genotyping master mix, 8.8 µL each of 10 µM wildtype and mutant probes, 39.6 µL each of 10 µM forward and reverse primers, and 17.6 µL of droplet stabilizing oil. Gently mix the dPCR reaction mixture by pipetting the full volume up and down 10–20 times. Do not vortex or centrifuge after dr…
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The authors have nothing to disclose.
Materials
| Raindance Consumable Kit | Bio-Rad | 20-04411 | Contains droplet-generating instrument chips, quantification instrument chips, PCR strip caps including high-speed caps, and droplet stabilizing oil |
| Raindance Sense Instrument | Bio-Rad | Quantification instrument | |
| Raindance Source Instrument | Bio-Rad | Droplet-generating instrument | |
| RainDrop Analyst II Software | RainDance Technologies | Analyst software used for Data Analysis | |
| Thermomixer C | Eppendorf | 14 285 562PM | |
| MiniAmp Thermal Cycler | Thermofisher Scientific | A37834 | |
| PCR 8 well strip tube caps | VWR | 10011-786 | |
| PCR 8 well strip tubes | Axygen | PCR-0208-C | |
| Q5 Hot Start High-Fidelity 2x Master Mix | New England Biolabs Inc | M0494S |
