Qiagen QIAcuity EG PCR Quick Start Guide

Sample to Insight__

January 2021

Quick-Start Protocol

QIAcuity® EG PCR Kit

This protocol is optimized for the quantification of DNA or cDNA targets using the QIAcuity EG

®

PCR Kit (cat. nos. 250111, 250112, and 250113) with the dsDNA-binding dye EvaGreen
singleplex reaction using QIAGEN’s QIAcuity instruments for digital PCR (dPCR).

The QIAcuity EG PCR Kit should be stored immediately upon receipt at –30 to –15°C in a constant­temperature freezer and protected from light. The QIAcuity EG PCR master mix can also be stored
protected from light at 2–8°C. Unless otherwise indicated on the label, the components are stable
for 6 months without showing any reduction in performance under these conditions.

Dedicated protocols for the various types of QIAGEN’s QIAcuity dPCR assays can be found in their
respective quick-start protocols and the

QIAcuity User Manual Extension: Application Guide

in a

.

Further information



QIAcuity User Manual Extension: Application Guide



QIAcuity User Manual

 Safety Data Sheets: www.qiagen.com/safety

 Technical assistance: support.qiagen.com

: www.qiagen.com/HB-2717

: www.qiagen.com/HB-2839

Notes before starting

 A fluorescent reference dye is provided as a component of the QIAcuity EG PCR master mix

for reliable detection of proper partition filling in the dPCR Nanoplates.

 For the highest efficiency in dPCR, amplicons should ideally be 60−150 bp in length.

Similar to qPCR, longer amplicons can be used as well; however, assay
performance might be impaired.

2

QIAcuity EG PCR Kit 01/2021

 Always start with the cycling conditions and primer concentrations specified in this protocol.
 The PCR must start with an initial incubation step of 2 min at 95°C to activate the

QuantiNova

 For ease of use, we recommend preparing a 10x or higher concentrated primer mix. A

®

DNA Polymerase in the master mix.

10x primer mix consists of 4 μM forward primer and 4 μM reverse primer in TE buffer with
low EDTA (0.1 mM).

 For 2-step RT-PCR, use the QuantiTect

®

Reverse Transcription Kit for the first step to

synthesize the cDNA.

Important: The QuantiNova Reverse Transcription Kit is not recommended.

The volume of the cDNA added (from the undiluted reverse-transcription reaction) should
not exceed 10% of the final PCR volume if using the QuantiTect Reverse Transcription Kit.
If using another RT kit, it should not exceed 5%.

Template DNA digestion

 DNA samples with an average length ≥20 kb (e.g., genomic DNA purified via spin column

with silica membrane, or salting out method) should be fragmented by restriction digestion
before partitioning. Enzymatic fragmentation of larger DNA ensures even distribution of
template throughout the QIAcuity Nanoplate, which in turn leads to accurate and precise
quantification.

 Fragmentation of DNA via restriction digest is particularly important when copy number

variation (CNV) analyses are performed, where multiple copies of a gene might be linked
in tandem. Restriction digestion is not required for highly fragmented DNA (e.g., FFPE DNA
or circulating DNA) or cDNA.

 Care should be taken to use enzymes that will not cut within the amplified sequence. For

QIAGEN’s CNV and mutation detection assays, appropriate information can be found at
geneglobe.qiagen.com.

 The following validated enzymes will digest DNA in 10 min at room temperature

(15−25°C) when added directly to the QIAcuity reaction mix at the indicated
concentrations.