Geode H14000 User Manual
15
non-specific hybridization of the primers/probe or competition phenomena, as well as good
estimated target concentration.
In addition, the efficiency of a single plex reaction should always be evaluated first before
proceeding to a multiplex one.
For optimal results, Stilla Technologies recommends the use of high-quality quenchers such as
Black Hole Quencher® (BHQ®). These quenchers absorb broadly, do not emit light, and thus
allow the use of multiple reporters with the same quencher (see
Table 1
for compatible
fluorophores).
Here are some guidelines for assay design:
•
Recommended primer and probe concentrations on the naica® system are 0.25 to 1 µM.
•
Before multiplexing, it is recommended to test each primer pair and the corresponding
probe in simplex reactions to verify PCR amplification.
•
Increasing the concentration of probes increases the basal fluorescence signal of the
negative droplets in the corresponding detection channel, and to a lesser extent in the
adjacent channels due to spill-over.
•
When multiplexing, depending on the primer and probe design, competition between the
different sequences can occur (for example, primer heterodimer formation or non-specific
annealing). High concentrations of primers and probes can aggravate these phenomena.
Thus, when combining several assays for high multiplexing, it is recommended to start
with low concentrations of primers for all assays (e.g., 0.25 µM), and increase the
concentrations gradually up to 1 µM if needed (for example to increase amplification
efficiency). (see
https://www.gene-pi.com/item/primers-and-probes-2/)
Note: When optimizing primer and probe concentrations for a given target, it can be beneficial to
determine the limiting factor first. For example, if the primers are the limiting factor, it is useless
to increase probe concentrations. Other important aspects to consider for assay optimization:
•
When designing primers and probes, try homogenizing the melting temperatures of all
the primers together and all the probes together. This will make it easier to pinpoint the
optimal elongation temperature for all targets.
•
When designing probes, short sequences of <20 nucleotides place the fluorophore and
quencher in close proximity and result in more efficient quenching. The result is increased
fluorescence differences between positive droplets (where probes are hydrolyzed) and
negative droplets (where probes remain intact).
ii.
Non-specific fluorescent intercalating dye: EvaGreen®
When setting up an experiment using EvaGreen®, it is important to remember that high
concentrations of any dsDNA initially present in the PCR mix will lead to higher basal
fluorescence. Moreover, although EvaGreen® has a higher affinity for dsDNA, the dye will also
be able to bind with lower affinity to any oligonucleotides present, thus also contributing to basal
fluorescence.
For optimal estimation of the sample concentration, use 0.2 up to 1,000 copies of template/µL of
final reaction.
Depending on the type of DNA to quantify and the intrinsic properties of the assay, it may be
necessary to assess the practical dynamic range.
