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CAUTION! Conical flask may be hot when removing from
the microwave oven. Ensure that protective heat gloves are
worn when handling hot flask.- Run a 5% agarose gel at 60 V for 150 min to determine
primer specificity and identify the best annealing tempera-
ture (see Note 9). - Non-template control must be used in all assays to ensure that
there is no cross contaminations (see Note 10). - Run a second set of gradient ddPCR to select the final anneal-
ing temperature. - Prepare reaction mixture according to the manufacturer’s pro-
tocol with consideration of pipetting error (see Note 11). - For example, according to the ddPCR supermix protocol, we
use a reaction mix. This contains 11 μL 2× ddPCR supermix
for probes, 1 μL of 1 μM control gene primer set, 1 μL of
1 μM target gene primer set, 1 μL of 2 U/μL restriction
enzyme (which does not cut the amplicon of both the target
gene and control gene) (see Note 12), and 30 ng of template.
DNase-free water is added to the reaction mix to top up to a
final volume of 22 μL. - Transfer 20 μL reaction volume into the sample section of a
microfluidic cartridge and subsequently with 70 μL of genera-
tion oil to the oil section. - Close the cartridge with a gasket and then place it into a drop-
let generator. - Carefully transfer all of the droplets into a 96-well plate (see
Note 13) and seal it with a pierceable foil heat seal.
3.4 ddPCR Gradient
Optimization
Fig. 2 Internal hybridization parameters section for design of probes
Katherine T. W. Lee et al.