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…

開示

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

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記事を引用
Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations. J. Vis. Exp. (Pending Publication), e20673, doi: (2023).

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