Bio-Rad ddPCR™ Supermix for Probes - Manual (Page 13)

QX200 Droplet Generator Instruction Manual | 5
Droplet Generation
2
2.1 Sample Preparation
Prepare the PCR reaction by combining 2x PCR supermix, 20x primers and
probe, and DNA sample. Mix by vortexing in short pulses; centrifuge briefly.
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The concentration of intact human genomic DNA should be <66 ng per
20 µl reaction. If using higher concentrations, digest DNA with a restriction
endonuclease that does not cut target or reference amplicons
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Use one of the PCR supermixes recommended in Table 1.2, as these contain
reagents required for droplet generation. Follow instructions in the product
inserts to prepare the samples for droplet generation
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Vortex the supermixes thoroughly to ensure homogeneity, as a concentration
gradient may form during –20°C storage. Alternatively, pipet up and down
>5 times to mix. Centrifuge briefly to collect contents at the bottom of the tube
before dispensing
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Thaw and equilibrate reaction components to room temperature. If the
sample is prone to thermal degradation, prepare the reaction mix on ice, but
equilibrate the reaction mix to room temperature (~3 min) before loading into
the DG8
™ cartridge for droplet formation
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Assemble reaction mixtures in vials or in 96-well PCR plates. The advantage
of using a PCR plate is that samples can be loaded into the DG8 cartridge
using an 8-channel pipet
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Use standard lab precautions to avoid contamination of the reaction mix and
sample: wear gloves, work in a clean area (such as a PCR hood), and use
clean pipets and low protein binding tubes