ddPCR™ Gene Expression EvaGreen Assays®

Catalog #Description

10031258
10031259
10031260

ddPCR Gene Expression EvaGreen® Assay, 200 reactions
ddPCR Gene Expression EvaGreen® Assay, 1,000 reactions
ddPCR Gene Expression EvaGreen® Assay, 2,500 reactions

For research purposes only.

Description
Droplet Digital™ PCR (ddPCR) Gene Expression EvaGreen®
Assays enable accurate quantification of small fold changes
for detecting targets that may be present at low levels or
substances in samples that can inhibit a PCR reaction.

Step 2: cDNA Amount to Include in ddPCR Reaction Mix

Ordering Information
Go to bio-rad.com/digital-assays to order ddPCR GeneExpression EvaGreen® Assays.

Step 3: ddPCR Reaction Mix Setup

Storage and Stability
The real-time PCR primer assays are stable for 12 months
when stored at 4°C. The 20x assay mix can be kept at –20°C
for long-term storage.
Kit Contents
The real-time PCR primer assay consists of unlabeled PCRprimer pairs for use with dye-based chemistry such as theQX200™ ddPCR EvaGreen® Supermix.
Reagents and Equipment■ iScript Advanced cDNA Synthesis Kit for RT-qPCR (catalog#1725037, #1725038)
■

■

■

■

 X200 ddPCR EvaGreen® Supermix (#1864033, #1864034,Q#1864035, #1864036)
 X200 Droplet Generator (#1864002) or Automated Droplet
QGenerator (#1864101)
 X200 Droplet Reader (#1864003) or QX600™ Droplet
QReader (#12013328)

Up to 6 μl of cDNA (not exceeding the equivalent of 50 nm
initial RNA) can be used per ddPCR reaction (20 μl finalvolume). Generally, the cDNA resulting from 1 ng of RNA
is sufficient for detection of most transcripts.
1.

Thaw all frozen reaction components to room temperature.
Mix thoroughly, then centrifuge briefly to collect solutions at
the bottom of tubes. Store the tubes on ice.

2.

Prepare the sample at room temperature according tothe recommendations in Table 1. If multiple samples
are to be assayed using the same target, prepare amaster reaction mix without sample template, dispense
equal aliquots into the reaction tubes, and add theresuspension of sample DNA.

Table 1. ddPCR reaction setup.
Component

Volume per
Reaction, µl

Final
Concentration

2x QX200 ddPCR EvaGreen® Supermix

101x

20x primers

1250 nM primers

cDNA with iScript Advanced cDNA
Synthesis Kit for RT-qPCR

Up to 6Up to 50 nm initial
RNARNase-/DNase-free water

Variable–Total volume20*–* For the Automated Droplet Generator, prepare 22 μl per well.

3.

Mix thoroughly by vortexing the tube. Centrifuge briefly
to ensure that all components are at the bottom of thereaction tube. Allow reaction tubes to equilibrate at room
temperature for about 3 minutes.

4.

Transfer the reaction mix from the reaction tubes to theappropriate ddPCR Cartridge as follows:PX1 PCR Plate Sealer (#1814000)

Refer to the instrument-specific manuals for ordering
information for other consumables (oils, cartridges, gaskets,plates, and seals).
ProtocolStep 1: cDNA Synthesis with iScript Advanced cDNA Synthesis Kit
for RT-qPCR

Make cDNA with iScript Advanced cDNA Synthesis Kit for
RT-qPCR according to the recommended protocol in theproduct insert (10042279).

— For the QX200 Droplet Generator, load 20 μl of eachreaction mix into a sample well of a DG8 Cartridge.
Follow subsequent instructions as specified inthe QX200 Droplet Generator Instruction Manual(10031907)
— For the Automated Droplet Generator, follow
instructions in the Automated Droplet Generator
Instruction Manual (10043138)

ddPCR Gene Expression EvaGreen® Assays

5.

Follow instructions for thermal cycling based on thedroplet generator used:

Visit bio-rad.com/DropletDigitalPCRAssays to learn more
about Bio-Rad’s Droplet Digital PCR solutions.

— For the QX200 Droplet Generator, carefully transfer
droplets into a clean 96-well plate. Seal the plate using
the PX1 PCR Plate Sealer at 180°C for 5 sec. Proceed
to thermal cycling (see Table 2)— For the Automated Droplet Generator, remove thedroplet plate containing ddPCR droplets from theAutomated Droplet Generator. Seal the plate using thePX1 PCR Plate Sealer at 180°C for 5 sec. Proceed tothermal cycling (see Table 2)
Table 2. Thermal cycling protocol.*Cycling Step

Temperature, °C TimeRamp
Rate

Numberof Cycles

Enzyme activation

95

5 min

~2°C/sec

1

Denaturation

96

30 sec40

Annealing/extension

58

1 min

40

Signal stabilization

45 min

190

5 min

4

Infinite

Hold (optional)

1–1

BIO-RAD, DDPCR, DROPLET DIGITAL, QX200, and QX600 are trademarks ofBio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are theproperty of their respective owner. © 2022 Bio-Rad Laboratories, Inc.
EvaGreen is a trademark of Biotium, Inc. Bio-Rad Laboratories, Inc. is licensed byBiotium, Inc. to sell reagents containing EvaGreen Dye for use in real-time PCR, forresearch purposes only.
Purchase of Digital PCR and/or Single-Cell NGS Sample Preparation products(the “Products”) from Bio-Rad Laboratories is subject to Bio-Rad Laboratories, Inc.
Standard Terms and Conditions of Sale, which can be accessed athttps://www.bio-rad.com/en-us/terms-conditions. Unless we expressly stateotherwise in additional Terms and Conditions, no rights are granted for you
to distribute or resell the Products. Unless we expressly state otherwise inadditional Terms and Conditions, no rights are granted for the development orcommercialization of diagnostic assays for use with the Products without a licensefrom Bio-Rad. The Products and/or their use are covered by U.S. and foreign
patents and/or pending patent applications owned by or under license to Bio-Rad
Laboratories, Inc. See https://www.bio-rad.com/en-us/trademarks.

* Use a heated lid set to 105°C and set the sample volume to 40 µl.

Data Acquisition and Analysis
Follow instructions for data acquisition and analysis based onthe droplet reader in use:■■ or the QX200 Droplet Reader, refer to the QX200 Droplet
FReader and QX Manager Software Standard Edition UserGuide (10000107223) or the QX200 Droplet Reader andQX Manager Software Regulatory Edition User Guide(10000107224)
 or the QX600 Droplet Reader, refer to the QX600 Droplet
FReader and QX Manager Software Standard EditionUser Guide (10000153877) or the QX600 Droplet Reader
and QX Manager Software Premium Edition User Guide(10000153878)

Other Recommendations
When running technical replicate wells, assemble a commonreaction mix (enough for twice as many wells as you plan torun) with all required components and sample template.
■■

Run at least 1 negative control
Runa positive control at a concentration similar to theunknown samples

Quality Control
ddPCR Assays are free of detectable DNase and RNase
activities. Stringent specifications are maintained to ensure
lot-to-lot consistency.

Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Drive, Hercules, CA 94547
510-741-1000

10000156270 Ver A (10041702)

22-03620622