ddPCR™ Supermix for Probes
Catalog #Description

1863026
18630101863027
1863028ddPCR Supermix for Probes, 2 ml (2 x 1 ml vials), 200 x 20 µl reactions
ddPCR Supermix for Probes, 5 ml (5 x 1 ml vials), 500 x 20 µl reactions
ddPCR Supermix for Probes, 25 ml (5 x 5 ml vials), 2,500 x 20 µl reactions
ddPCR Supermix for Probes, 50 ml (10 x 5 ml vials), 5,000 x 20 µl reactions

For research purposes only.

Description
ddPCR Supermix for Probes is a 2x concentrated, ready-touse reaction cocktail containing all components — exceptprimers, probe(s), and template — required for probe-based
Droplet Digital™ PCR (ddPCR). The mixture delivers maximum
target specificity and fluorescence amplitude and minimum
droplet variability to ensure precise target quantification.
The hot-start feature of the polymerase in the supermix
enables partitioning of sample into droplets while keeping
the enzyme inactive at ambient conditions. The supermix has
been optimized to support the amplification and detectionof DNA targets using commercially available probe-based
assays and is suitable for use with uracil N-glycosylase (UNG)
for PCR decontamination. UNG may be purchased from alicensed supplier.
Storage and Stability
ddPCR Supermix for Probes is stable at –20°C through theexpiration date printed on the label. Once thawed, it canbe stored at 4°C for up to 2 weeks. Repeated freezing andthawing of the supermix is not recommended.
Quality Control
ddPCR Supermix for Probes is free of contaminating DNaseand RNase. Stringent specifications are maintained to ensure
lot-to-lot consistency.
Recommendations for Optimal Results■ Follow general guidelines and recommendations for Droplet
Digital PCR (refer to the Droplet Digital PCR Applications
Guide, bulletin 6407)■

 he concentration of intact human genomic DNA shouldTbe ≤66 ng per 20 μl reaction. If using higher concentrations,digest DNA with a restriction endonuclease (see guidelinesin DNA Digestion section)

Required Equipment
The QX200™ Droplet Digital PCR System (catalog #1864001),
QX200 AutoDG™ Droplet Digital PCR System (#1864100),
QX600™ Droplet Digital PCR System (#17007769),
QX600 AutoDG Droplet Digital PCR System (#17008371),
or QX ONE™ Droplet Digital PCR System (#12006536)
is required.
Refer to the QX200 Droplet Reader and QX Manager SoftwareStandard Edition User Guide and QX200 Droplet Generator
Instruction Manual (10000107223 and 10031907, respectively),
the Automated Droplet Generator Instruction Manual(10043138), or the QX ONE Droplet Digital PCR System

and QX ONE Software User Guide (10000116512) for
ordering information about consumables, such as oils,cartridges, gaskets, plates, and seals.
Reaction Setup
1. Thaw all components to room temperature. Mix thoroughly
by vortexing each tube to ensure homogeneity
because a concentration gradient may form during–20°C storage. Centrifuge briefly to collect contents
at the bottom of the tubes.
2.

Prepare samples at the desired concentration before
setting up the reaction mix.

3.

Prepare the reaction mix for the number of reactions
needed according to the guidelines in Table 1. Assemble
all required components except the sample, dispense
equal aliquots into each reaction tube, and add thesample to each reaction tube as the final step.

Table 1. Preparation of the reaction mix.
Volume per
Reaction, µl

Final
Concentration

2x ddPCR Supermix for Probes

10

1x

20x target primers/probe(FAM, Cy5, or ROX)

1900 nM/250 nM

20x target primers/probe(HEX/VIC, Cy5.5, or ATTO 590)

1

900 nM/250 nM

Diluted restriction enzyme(see DNA Digestion section)

1

Variable

SampleVariable

Up to 330 ng*

RNase-/DNase-free water

Variable—20—

Component

Total volume*** Sample concentrations >66 ng per reaction and certain applications may
require restriction digestion for optimal target detection. If digestion is not
required, prepare the ddPCR reaction mix without the diluted
restriction enzyme.
** For the Automated Droplet Generator, prepare 22 μl per reaction.

4.

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

5.

Transfer the reaction mix from the reaction tubes to theappropriate ddPCR Cartridge as follows:■ or the QX600 or QX200 Droplet Digital PCR System,
Fload 20 μl of each reaction mix into a sample well of a
DG8 Cartridge. Follow subsequent instructions as
specified in the QX200 Droplet Generator Instruction
Manual (10031907)

ddPCR Supermix for Probes■■

 or the QX600 or QX200 AutoDG Droplet Digital PCR
FSystem, follow instructions in the Automated Droplet
Generator Instruction Manual (10043138)
 or the QX ONE Droplet Digital PCR System, loadF
20 μl of each reaction mix into a sample well of a
GCR96 Cartridge. Follow subsequent instructions as
specified in the QX ONE Droplet Digital PCR System
and QX ONE Software User Guide (10000116512)

DNA Digestion (recommended)
DNA fragmentation by restriction digestion prior to droplet
generation enables optimal accuracy by separating tandem
gene copies, reducing sample viscosity, and improving template
accessibility for input samples >66 ng per well. Choose arestriction endonuclease that does not cut either the target orreference amplicon and that is insensitive to methylation.
Four-base cutters and high-fidelity enzymes are preferred.
Two strategies may be used to perform restriction digestionof DNA samples: digestion directly in the ddPCR reaction during
setup or conventional digestion prior to Droplet Digital PCR.

■

■

■

■

■

 ilute the restriction enzyme using the recommendedD
diluent buffer according to the manufacturer’s instructions
and add 1 µl to the ddPCR reaction as outlined in Table 1
 pproximately 2–5 units of restriction enzyme per 20 µl
A
ddPCR reaction are recommended
 he addition of restriction enzyme buffers with high saltT
can inhibit Droplet Digital PCR and should be avoided

Temperature,°C

Time

Numberof Cycles

Hold (QX ONE ddPCRSystem only)

25

3 min

1

Enzyme activation95

10 min1

Cycling StepDenaturation

94

30 sec40

Annealing/extension

60

1 min**

40

Enzyme deactivation98

10 min1QX600 or QX200
ddPCR System

430 min1

QX ONE ddPCRSystem251 min1Hold*	For the PTC Tempo Deepwell Thermal Cycler or C1000 Touch Thermal Cycler
with 96–Deep Well Reaction Module, use a heated lid set to 105°C and set
the sample volume to 40 μl.
** Check/adjust ramp rate settings to ~2°C/sec.

Data Acquisition and Analysis
Follow instructions based on the system in use:■

 aeIII, MseI, AluI, HindIII, and CviQI have been observedH
to work well for digestions in ddPCR reactions

Digestion Prior to Droplet Digital PCR
■

■■■

■

■■

 se 5–10 enzyme units per microgram DNA andU
10–20 enzyme units per microgram genomic DNA■Incubate the reaction for 1 hr at the temperature
recommended for the restriction enzyme

 or the QX600 Droplet Digital PCR System and theF
QX600 AutoDG Droplet Digital PCR System, refer tothe QX600 Droplet Reader and QX Manager SoftwareStandard Edition User Guide (10000153877)
 or the QX200 Droplet Digital PCR System and theF
QX200 AutoDG Droplet Digital PCR System, refer tothe QX200 Droplet Reader and QX Manager SoftwareStandard Edition User Guide (10000107223)

DNA purification is not necessary after restriction digestion or the QX ONE Droplet Digital PCR System, referF
to the QX ONE Droplet Digital PCR System and
QX ONE Software User Guide (10000116512) and the
QX ONE Software User Guide for Standard Edition(10000116655) or Regulatory Edition (10000116656)

 se a minimum 10-fold dilution of the digest to reduce theUsalt content of the sample in the ddPCR reaction

Visit bio-rad.com/ddPCRsmxProbes for more information.

 eat inactivation is not required, but can be consideredH
if long-term storage is required; do not heat inactivate
above 65°C

Store digested DNA at –20°C or below
 repare the ddPCR reaction mix, as outlined in Table 1,P
without the diluted restriction enzyme

Thermal Cycling Conditions
Follow instructions based on the system in use:■ or the QX ONE Droplet Digital PCR System, useFappropriate thermal cycling conditions as specified
in Table 2 during plate setup. Refer to the QX ONE
Droplet Digital PCR System and QX ONE Software
User Guide (10000116512) for plate setup instructions

Table 2. Thermal cycling conditions.*

Digestion in ddPCR Reaction■

 or the QX600 or QX200 AutoDG Droplet Digital PCR
FSystem, seal the 96-well plate using the PX1 PCR Plate
Sealer at 180°C for 5 sec. Proceed to thermal cycling(see Table 2)

 or the QX600 or QX200 Droplet Digital PCR System,F
carefully transfer droplets into a clean 96-well plate. Seal theplate using the PX1 PCR Plate Sealer (#1814000) at 180ºCfor 5 sec. Proceed to thermal cycling (see Table 2)

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510-741-1000■BIO-RAD, AUTODG, DDPCR, DROPLET DIGITAL, QX200, QX600, and QX ONE are
trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks usedherein are the property of their respective owner. © 2023 Bio-Rad Laboratories, Inc.
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 at https://www.
bio-rad.com/en-us/terms-conditions. Unless we expressly state otherwise inadditional Terms and Conditions, no rights are granted for you to distribute orresell the Products. Unless we expressly state otherwise in additional Terms andConditions, ​no rights are granted for the development or commercialization ofdiagnostic assays for use with the Products without a license from Bio-Rad. It is theuser’s obligation to obtain a commercial license from Bio-Rad for (i) all commercial
uses (not just diagnostic uses) and (ii) sale of assays for use on Bio-Rad’s dPCR andddSEQ instruments. The Products and/or their use are covered by U.S.​and foreign
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10000162843 Ver B (10026235)

23-03590423