Bio-Rad QX100™ Droplet Digital™ PCR System - Manual (Page 11)

QX100 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, and 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 either the ddPCR ™ supermix for probes or the One-Step RT-PCR kit
for probes, which contain reagents required for droplet generation. Follow
the 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 minutes) 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, clean pipets, work in a clean area such as a PCR hood,
and use low protein binding tubes