Thermo Scientific Applied Biosystems QuantStudio - Recommended DNA Volume and Concentration; Prepare the Dpcr Reaction MIX for each Sample

The recommended DNA volume in the following reaction is based on a human gDNA
sample at 10 ng/µL concentration (nal concentration in the reaction is ~3.45 ng/μL),
with the target sequence present at two copies per diploid genome. This
recommended volume will vary depending upon species, sample type, and sample
concentration. For best results, we recommend adding sufcient DNA such that the
concentration of target sequence in the nal reaction is between 200 and
2,000 copies/µL.
Prepare the PCR reactions for loading on the QuantStudio
™
3D Digital PCR
20K Chip v2.
1.
Thaw the following at room temperature, and ensure that the tubes are at room
temperature before using:
•
QuantStudio
™
3D Digital PCR Master Mix v2
•
TaqMan
™
Assay(s)
2.
Prepare the genomic DNA sample:
a.
Dilute the DNA sample as needed so that the concentration of target
sequence in the nal reaction is between 200 and 2,000 copies/µL.
b.
Vortex, then briey centrifuge the DNA sample.
c.
Using a permanent marker, label a 0.5- or 1.5-mL reaction tube with the
sample name.
3.
When the master mix has warmed to room temperature, gently invert the tube
10 times (or gently vortex on low-medium speed).
4.
In a 0.5- or 1.5-mL low-bind tube, prepare the following dPCR reaction mix at
room temperature. Scale the volumes appropriately, depending on the number
of samples.
Table 1 dPCR reaction mix—example amounts
The example amounts assume that you are running two chips per human gDNA sample at 10 ng/µL.
Item
Amount
1 sample/
2 chips
[1]
Stock Final
Master mix 17.4 μL 2X 1X
TaqMan
™
Assay(s), 20X
(primer/probe mix)
1.7 μL 20X 1X
Diluted genomic DNA
[2]
3.5 μL 10 ng/µL 1 ng/µL
Water 12.2 μL — —
Total volume 34.8 μL — —
Volume to load per chip 14.5 μL — —
[1]
Volumes include 20% excess to compensate for volume loss from pipetting.
[2]
Depending on sample source, required input DNA amount may vary.
Recommended
DNA volume
and
concentration
Prepare the
dPCR reaction
mix for each
sample
Chapter 3 Prepare the digital PCR reaction and load the chips
Prepare the digital PCR reactions
3
QuantStudio
™
3D Digital PCR System User Guide
23