ddPCR™ Supermix for Probes (No dUTP)
Catalog #Description

1863023
1863024
1863025

ddPCR Supermix for Probes (No dUTP), 2 ml (2 x 1 ml vials), 200 x 20 µl reactions
ddPCR Supermix for Probes (No dUTP), 5 ml (5 x 1 ml vials), 500 x 20 µl reactions
ddPCR Supermix for Probes (No dUTP), 25 ml (5 x 5 ml vials), 2,500 x 20 µl reactions

For research purposes only.

Description
ddPCR Supermix for Probes (No dUTP) is a 2x concentrated,
ready-to-use reaction cocktail containing all components —except primers, probe(s), and template — required forprobe-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 theenzyme inactive at ambient conditions. The supermix has been
optimized to support the amplification and detection of DNA
targets using commercially available probe-based assays, andis also suitable for applications such as library quantification andsample preparation for next-generation sequencing.
Storage and Stability
ddPCR Supermix for Probes (No dUTP) is stable at –20°C
through the expiration date printed on the label. Once thawed,
it can be stored at 4°C for up to 2 weeks. Repeated freezing
and thawing of the supermix is not recommended.

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 ofthe tubes.
2. P repare 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 the sample
to each reaction tube as the final step.
Table 1. Preparation of the reaction mix.
Component

Volume per Reaction, µl Final Concentration

2x ddPCR Supermix forProbes (No dUTP)

101x

Quality Control
ddPCR Supermix for Probes (No dUTP) is free of contaminating
DNase and RNase. Stringent specifications are maintained
to ensure lot-to-lot consistency.

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

1900 nM/250 nM

20x target primers/probe(HEX/VIC, Cy5.5, or ATTO590)

1

900 nM/250 nM

Recommendations for Optimal Results■ Follow general guidelines and recommendations forDroplet Digital PCR (refer to the Droplet Digital PCR
Applications Guide, bulletin 6407)

Diluted restriction enzyme(see DNA Digestion section)

1Variable

SampleVariable

Up to 330 ng*

RNase-/DNase-free water

Variable—20—■ he concentration of intact human genomic DNA
Tshould be ≤66 ng per 20 μl reaction. If using higher
concentrations, digest DNA with a restriction endonuclease(see guidelines in 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
Software Standard 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

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. M
 ix thoroughly by vortexing the tubes. Centrifuge briefly
to ensure that all components are at the bottom of thereaction tubes. Allow reaction tubes to equilibrate at room
temperature for about 3 minutes.
5. T
 ransfer 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 ofa DG8 Cartridge. Follow subsequent instructions as
specified in the QX200 Droplet Generator Instruction
Manual (10031907)ddPCR Supermix for Probes (No dUTP)■■

 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, load
F
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 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 a
restriction endonuclease that does not cut either the target
or reference 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.
Digestion in ddPCR Reaction
■■

■

■

 ilute the restriction enzyme using the recommended
Ddiluent 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
AddPCR reaction are recommended
 he addition of restriction enzyme buffers with high salt canTinhibit Droplet Digital PCR and should be avoided
 aeIII, MseI, AluI, HindIII, and CviQI have been observed toH
work well for digestions in ddPCR reactions

■

■

■

Temperature,°C

Time

Numberof Cycles

Hold (QX ONE ddPCRSystem only)

253 min1

Enzyme activation95

10 min1Cycling StepDenaturation

9430 sec40

Annealing/extension

60

1 min**

40

Enzyme deactivation98

10 min1QX600 or QX200
ddPCR System

430 min

1QX ONE ddPCR
System251 min

1Hold*	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:
■■■

■■■

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

 se a minimum 10-fold dilution of the digest to reduceU
the salt content of the sample in the ddPCR reaction
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:■■ eat inactivation is not required, but can be considered if longH
term storage is required; do not heat inactivate above 65°C
DNA purification is not necessary after restriction digestion or the QX600 or QX200 Droplet Digital PCR System,
Fafter droplet generation with the QX200 Droplet Generator,
carefully transfer droplets into a clean 96-well plate. Seal theplate using the PX1 PCR Plate Sealer (#1814000) at 180°C
for 5 sec. Proceed to thermal cycling (see Table 2)

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

 or the QX ONE Droplet Digital PCR System, thermalFcycling is integrated into and sequentially performed by thesystem itself. Hence, no additional equipment or sample
handling is required for this step. Refer to the QX ONEDroplet Digital PCR System and QX ONE Software User
Guide (10000116512) for plate setup instructions. Useappropriate thermal cycling conditions as specified in Table 2Table 2. Thermal cycling conditions.*

Digestion Prior to Droplet Digital PCR■

 or the QX600 or QX200 AutoDG Droplet Digital PCR
FSystem, remove the droplet plate containing ddPCR dropletsfrom the Automated Droplet Generator. Seal the plate using
the PX1 PCR Plate Sealer at 180°C for 5 sec. Proceed tothermal cycling (see Table 2)■

 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)
 the QX ONE Droplet Digital PCR System, refer
Forto the QX ONE Droplet Digital PCR System andQX ONE Software User Guide (10000116512) and the
QX ONE Software User Guide for Standard Edition(10000116655) or Regulatory Edition (10000116656)

Visit bio-rad.com/ddPCRsmxNodUTP for more information.
BIO-RAD, AUTODG, DDPCR, DROPLET DIGITAL, QX200, QX600, and QX ONE are trademarks ofBio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein 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 Conditionsof Sale, which can be accessed at https://www.bio-rad.com/en-us/terms-conditions. Unless
we expressly state otherwise in additional Terms and Conditions, no rights are granted for you
to distribute or resell the Products. Unless we expressly state otherwise in additional Terms andConditions, n​ o rights are granted for the development or commercialization of diagnostic assays
for use with the Products without a license from Bio-Rad. It is the user’s obligation to obtain acommercial license from Bio-Rad for (i) all commercial uses (not just diagnostic uses) and (ii) saleof assays for use on Bio-Rad’s dPCR and ddSEQ instruments. The Products and/or their use arecovered 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.

10000162844 Ver B (10026868)

23-03560423