ddPCR™ Mutation Detection Assays, Validated
Catalog #Description

10031246
10031247
10031248
10031249
10031250
10031251

ddPCR Mutation Detection Assay, Validated (FAM), 200 x 20 μl reactions
ddPCR Mutation Detection Assay, Validated (FAM), 1,000 x 20 μl reactions
ddPCR Mutation Detection Assay, Validated (FAM), 2,500 x 20 μl reactions
ddPCR Mutation Detection Assay, Validated (HEX), 200 x 20 μl reactions
ddPCR Mutation Detection Assay, Validated (HEX), 1,000 x 20 μl reactions
ddPCR Mutation Detection Assay, Validated (HEX), 2,500 x 20 μl reactions

For research purposes only.

Description
ddPCR Mutation Detection Assays, Validated, are expertly
designed specifically for Droplet Digital™ PCR (ddPCR) using
proprietary computational algorithms. These assays have been
wet-lab validated by Bio-Rad™ Laboratories, Inc. Validation
data are available for review and download from the assay
page on the Bio-Rad website. Reference assays with thecomplementary fluorophores can be purchased separately.

Refer to the QX200 Droplet Generator Instruction Manual(10031907), QX ONE Droplet Digital PCR System andQX ONE Software User Guide (10000116512), or AutomatedDroplet Generator Instruction Manual (10043138) for ordering
information on consumables such as oils, cartridges, gaskets,plates, and seals. Refer to Table 1 for fluorophore compatibility
with instruments.

Ordering Information
Visit bio-rad.com/digital-assays to order ddPCR Mutation
Detection Assays.

QX200 Droplet Reader

QX ONE ddPCR System QX600 Droplet Reader

FAM

FAM

FAM

HEX

HEX

HEX

Storage and Stability
ddPCR Mutation Detection Assays are stable for 12 months
when stored at 4°C protected from light. The 20x assay mix
can be kept at –20°C for long-term storage.
Kit Contents
The ddPCR Mutation Detection Assay is a 20x concentrated,ready-to-use primer-probe mix. Each kit comes with 200,1,000, or 2,500 μl of the 20x assay mix (9 μM primers and5 μM probe), sufficient for 200, 1,000, or 2,500 x 20 μl
reactions, respectively.
Reagents and Equipment
For assays using the QX200™ Droplet Generator (catalog#1864002) or Automated Droplet Generator (#1864101):
■

■

■

■

■

Restriction Enzyme Digestion of Sample DNA
DNA fragmentation by restriction digestion prior to droplet
generation enables optimal accuracy by separating tandem
gene copies, reducing sample viscosity, and improving
template accessibility. Two strategies may be used to perform
restriction digestion of DNA samples: digestion directly in theddPCR reaction during setup (recommended) or conventional
digestion prior to Droplet Digital PCR.
Digestion Directly in ddPCR Reaction■■■

For 1–2 targets, ddPCR Supermix for Probes (No dUTP)(#1863023, 1863024, 1863025) is recommended
For >2 targets, ddPCR Multiplex Supermix (#12005909,12005910, 12005911) is recommended
QX200 Droplet Reader (#1864003) or QX600™ Droplet
Reader (#12013328)
PX1 PCR Plate Sealer (#1814000)

For assays using the QX ONE™ Droplet Digital PCR System(#12006536):■

Table 1. Fluorophore compatibility.

ddPCR Multiplex Supermix (#12005909, 12005910,12005911)
PX1 PCR Plate Sealer (#1814000)■■

Efficient digestion of sample DNA can be achieved by direct
addition of restriction enzyme to the ddPCR reaction
2–5 units of restriction enzyme per ddPCR reaction
are recommended
Dilute the restriction enzyme using the recommended
diluent buffer according to the manufacturer’s instructions,and then add 1 μl to the ddPCR reaction according to theguidelines in Table 2Reactions can be set up at room temperature; no additional
incubation time is required
The addition of restriction enzyme buffers with highsalt content can inhibit Droplet Digital PCR and should
be avoided

ddPCR Mutation Detection Assays, Validated

Digestion Prior to Droplet Digital PCR
■

■

■

■

■

■

■

Table 2. Preparation of the reaction mix.

Restriction enzyme digestion can be carried out asa separate reaction before ddPCR reaction setup
Use 10–20 units of restriction enzyme per microgramof genomic DNA
Incubate the reaction for 1 hr at the temperature
recommended for the restriction enzyme
Heat inactivation is not required but can be considered
if long-term storage is required; do not heat inactivate
above 65°C

3.

4.

DNA purification is not necessary after restriction digestion

10

1x

20x target primers/probe (FAM)

1

1x***

20x reference primers/probe (HEX)

1

1x***

Restriction enzyme,
diluted*1

2–5 U/reaction

DNA sample or water

Variable

50 fg–100 ng†

Total volume

20**–This component should be replaced by water if digestion is performed priorto Droplet Digital PCR.
** For the Automated Droplet Generator, prepare 22 μl per well.
*** 450 nM primers/250 nM each probe.
†For most assays, up to 130 ng of DNA containing mutant DNA at >2%
fractional abundance is expected to perform adequately. Input may belowered if cluster separation is not adequate.

Use a minimum tenfold dilution of the digest to reduce thesalt content of the sample in Droplet Digital PCRStore digested DNA at –20°C or below

Prepare samples at the desired concentration before
setting up the reaction mix according to the guidelines
in Table 2. If multiple samples are to be assayed using
the same target and reference duplex, prepare a master
reaction mix without sample template, dispense equal
aliquots into the reaction tubes, and add the sample
template to each reaction tube as the final step.

5.

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

6.

Transfer the reaction mix from the reaction tubes tothe appropriate ddPCR Cartridge as follows:– For the QX200 Droplet Generator, load 20 μl ofeach reaction mix into a sample well of a DG8Cartridge. Follow subsequent instructions as
specified in the QX200 Droplet Generator
Instruction Manual (10031907)– For the Automated Droplet Generator, follow
instructions in the Automated Droplet Generator
Instruction Manual (10043138)

Prepare a negative control: at least one well containing
only wild-type template at a concentration similar to theconcentration of unknown samples.

– For the QX ONE ddPCR System, load 20 μl of eachreaction 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)

Prepare a positive control: at least one well with a mixof 7 ng of mutant DNA in a background of 130 ng ofwild-type DNA.

© 2022 Bio-Rad Laboratories, Inc.

Volume per Reaction, μl Final Concentration

2x ddPCR Supermix
for Probes (No dUTP)*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 thebottom of each tube and store protected from light.
2.

Component

210000156271

ddPCR Mutation Detection Assays, Validated

Thermal Cycling Conditions
Follow instructions for thermal cycling based on the droplet
generator used:■■■

Data Acquisition and Analysis
Follow instructions for data acquisition and analysis basedon the droplet reader in use:

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 3)■

For the Automated Droplet Generator, remove the droplet
plate containing ddPCR droplets from the AutomatedDroplet Generator. Seal the plate using the PX1 PCR PlateSealer at 180°C for 5 sec. Proceed to thermal cycling(see Table 3)■

For the QX ONE ddPCR System, thermal cycling is integrated
into and sequentially performed by the system itself. Hence,no additional equipment or sample handling is required forthis step. Refer to the QX ONE Droplet Digital PCR System
and QX ONE Software User Guide (10000116512) for plate
setup instructions. Use appropriate thermal cycling conditions
as specified in Table 3.

■

For the QX200 Droplet Reader, refer to the QX200 Droplet
Reader and QX Manager Software Standard Edition UserGuide (10000107223) or the QX200 Droplet Reader and
QX Manager Software Regulatory Edition User Guide(10000107224)
For the QX600 Droplet Reader, refer to the QX600 Droplet
Reader and QX Manager Software Standard Edition
User Guide (10000153877) or the QX600 Droplet Reader
and QX Manager Software Premium Edition User Guide(10000153878)
For the QX ONE ddPCR System, refer to the QX ONE Droplet
Digital PCR System and QX ONE Software User Guide(10000116512) and the QX ONE Software Standard
Edition or Regulatory Edition User Guide (10000116655or 10000116656, respectively)

Table 3. Thermal cycling conditions.*
Cycling Step

Temperature, °C

Time

Numberof Cycles

Hold (QX ONE ddPCR
System only)

25

3 min

1

Enzyme activation95

10 min1

Denaturation

94

30 sec40

Annealing/extension

55

1 min**

40

Enzyme deactivation98

10 min1Hold4Infinite

1

QX ONE ddPCR 25
System (required)

1 min

1QX200 or QX600
ddPCR System(optional)* For the C1000 Touch Thermal Cycler, 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© 2022 Bio-Rad Laboratories, Inc.

310000156271

ddPCR Mutation Detection Assays, Validated

1016006
BIO-RAD, DDPCR, DROPLET DIGITAL, QX200, QX600, and QX ONE are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are theproperty of their respective owner. © 2022 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. StandardTerms and Conditions of Sale, which can be accessed at https://www.bio-rad.com/en-us/terms-conditions. Unless we expressly state otherwise in additional Terms andConditions, no rights are granted for you to distribute or resell the Products. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted forthe development or commercialization of diagnostic assays for use with the Products without a license from Bio-Rad. The Products and/or their use are covered by U.S. andforeign 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.

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

10000156271 Ver A (10033487)

22-0358

0622