Qiagen QIAcuity 911000 User Manual page 185

1. Prepare your master mix according to your reaction setup. To prepare the reaction mix
without sample, the QIAcuity PCR Master Mix has to be mixed with primers, RNase-free water
and optionally restrictions enzyme and probes according to the kit manual. The final volume
depends on the QIAcuity Nanoplate which is used (refer to table 19).
Note: To prevent non-homogeneous reaction mixes, the set up in a standard PCR pre-plate is
required. The calculated reagent volumes have to be pipetted into the PCR pre-plate, and then
the sample has to be added accordingly. For homogenous mixing of reaction mix, the pre-
plate has to be sealed, shortly vortexed and briefly centrifuged.
Note: Enzymatic fragmentation of DNA larger than 20 kilobase ensures even distribution of
template throughout the QIAcuity Nanoplate, which in turn leads to accurate and precise
quantification. Therefore, adding a restriction enzyme depends on the template used. In case
of enzymatic fragmentation using the recommended restriction enzymes, the pre-plate has to
be incubated at RT for 10 minutes. Longer incubation doesn't lead to unspecific restriction and
therefore has no impact on the result. Refer to the Application guide on www.qiagen.com for
the recommended restriction enzymes.
Important: Do not pipet master mix and sample separately into the Nanoplate as this will lead
to insufficient mixing.
2. Pipet each reaction mix from the pre-plate into a well of the Nanoplate. If possible, use an
electric one-channel pipette. To ensure bubble-free pipetting, we recommend to pipette 39µl
for Nanoplate 26K 24-well, and 11µl for Nanoplate 8.5K 96/24-well of your prepared
reaction mix to the bottom of the respective input well of the Nanoplate. Ensure not to pipet
into the output well instead of the input well.
Note: Do not centrifuge the Nanoplate as this will lead to pre-priming and insufficient filling of
the wells.
Note: Do not vortex the Nanoplate as this will lead insufficient filling of the wells.
3. Apply the plate seal that comes with the Nanoplates as follows to ensure good filling of the
wells and to prevent evaporation and contamination:
The stiff plate seal consists of a plate seal and 2 protective foils. The 3 layered foil should not
be folded. Remove the bottom white protective foil carefully, center and align the plate seal
(still containing the upper protective foil) with the lower edge of the colored frame of row H.
The foil should not overlap on any side more than 1mm, otherwise the Nanoplate might not
be processed by the instrument. In case the plate seal is incorrectly placed or the seal does
not cover some parts of the Nanoplate, carefully remove this seal and repeat sealing step with
a new one. Correct sealing of the Nanoplate prevents samples from not being fully processed.
4. After correct placing, the plate seal has to be fixed with the Nanoplate roller in the horizontal
and vertical direction.
QIAcuity User Manual 07/2020 185