ddPCR™ Mutation Detection Assays
Catalog #Description

10042964
10042965
10042966
10042967
10042968
10042969
12008220
12008221
12008222
12008223
12008224
12008225
12016622
12016623
12016631
12016592
12016632
12016613

ddPCR Mutation Detection Assay (FAM), 200 x 20 μl reactions
ddPCR Mutation Detection Assay (FAM), 1,000 x 20 μl reactions
ddPCR Mutation Detection Assay (FAM), 2,500 x 20 μl reactions
ddPCR Mutation Detection Assay (HEX), 200 x 20 μl reactions
ddPCR Mutation Detection Assay (HEX), 1,000 x 20 μl reactionsddPCR Mutation Detection Assay (HEX), 2,500 x 20 μl reactions
ddPCR Mutation Detection Assay (Cy5), 200 x 20 μl reactions
ddPCR Mutation Detection Assay (Cy5), 1,000 x 20 μl reactions
ddPCR Mutation Detection Assay (Cy5), 2,500 x 20 μl reactions
ddPCR Mutation Detection Assay (Cy5.5), 200 x 20 μl reactions
ddPCR Mutation Detection Assay (Cy5.5), 1,000 x 20 μl reactions
ddPCR Mutation Detection Assay (Cy5.5), 2,500 x 20 μl reactions
ddPCR Mutation Detection Assay (ROX), 200 x 20 μl reactions
ddPCR Mutation Detection Assay (ROX), 1,000 x 20 μl reactions
ddPCR Mutation Detection Assay (ROX), 2,500 x 20 μl reactions
ddPCR Mutation Detection Assay (ATTO 590), 200 x 20 μl reactions
ddPCR Mutation Detection Assay (ATTO 590), 1,000 x 20 μl reactions
ddPCR Mutation Detection Assay (ATTO 590), 2,500 x 20 μl reactions

For research purposes only.

Description
ddPCR Mutation Detection Assays are expertly designed
specifically for Droplet Digital™ PCR (ddPCR) using proprietarycomputational algorithms. These assays have not been wet-lab
validated by Bio-Rad™ Laboratories, Inc. Performance of theseassays should be validated prior to use. Reference assays withthe complementary fluorophore can be purchased separately.
Ordering Information
Visit bio-rad.com/digital-assays to order ddPCR Mutation
Detection Assays.
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):
■

■

■

■

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™ DropletReader (#12013328)
PX1 PCR Plate Sealer (#1814000)

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

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

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. See Table 1 for fluorophore compatibility
with instruments.
Table 1. Fluorophore compatibility.
QX200 Droplet Reader

QX ONE ddPCR System

QX600 Droplet Reader

FAMFAM

FAM

HEX

HEX

HEX

Cy5Cy5Cy5.5

Cy5.5ROX
ATTO 590

Determination of Optimal Annealing Temperature
Newly designed ddPCR Mutation Detection Assays should
be run across a thermal gradient (50–60°C), to determine theannealing/extension temperature that optimizes separation
between positive and negative droplets while minimizing rain
(droplets that fall between the major positive and negative
populations). If possible, an annealing/extension temperaturethat optimizes performance of both target and reference
assays should be selected.

ddPCR Mutation Detection Assays

■

■

■

■

■

■

Using a sample containing both mutant and wild-type alleles
as template, prepare reaction mix for at least 8 wells (1 column)
according to the guidelines in the Reaction Setup section

ddPCR reaction during setup (recommended) or conventional
digestion prior to Droplet Digital PCR.
Digestion Directly in ddPCR Reaction

For optimal performance, follow recommendations in theRestriction Enzyme Digestion of Sample DNA and Reaction
Setup sections■■

After droplet generation, proceed to thermal cycling on aC1000 Touch Thermal Cycler with 96–Deep Well Reaction
Module (1851197). Use an annealing temperature gradient
as described in Table 2■

Optimum annealing temperature range is determined basedon the separation between four clusters (Figure 1)■

For more information, see Rare Mutation Detection Best
Practices Guidelines (bulletin 6628)■

Run several no template control (NTC) wells

Channel 1 amplitude

20,000■10,000Droplets contain only
mutant template

■

5,000

Droplets containno template
Droplets contain only
wild-type template

■

00

2,000

4,000

6,0008,000
Channel 2 amplitude

10,00012,000Fig. 1. Two-dimensional scatter plot demonstrating the four clusters
obtained with a mutant and wild-type allele.

Cycling StepTemperature, °C

Time

Numberof Cycles

Hold (QX ONE ddPCRSystem only)

25

3 min

1

Enzyme activation95

10 min1

Denaturation

94

30 sec40

Annealing/extension~50–601 min**

40

Enzyme deactivation98

10 min1

Hold

4

Infinite1

QX ONE ddPCR 25
System (required)

1 min1■■

Table 2. Thermal gradient cycling conditions for determination ofoptimal annealing/extension temperature.*QX200 or QX600
ddPCR System(optional)■■

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 3
Reactions 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
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
DNA purification is not necessary after restriction digestion
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

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.

Prepare samples at the desired concentration before
setting up the reaction mix according to the guidelines
in Table 3. 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.

3.

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

4.

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.

* 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

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 the© 2022 Bio-Rad Laboratories, Inc.

2–5 units of restriction enzyme per ddPCR reaction arerecommended

Digestion Prior to Droplet Digital PCR

Droplets containboth templates

15,000Efficient digestion of sample DNA can be achieved by direct
addition of restriction enzyme to the ddPCR reaction

210000156273

ddPCR Mutation Detection Assays

Table 3. Preparation of the reaction mix.

■Component

Volume per Reaction, μl

Final Concentration

2x ddPCR Supermix
for Probes (No dUTP)

101x20x target primers/
1probe (FAM, Cy5 or ROX)

1x***

20x reference primers/probe (HEX, Cy5.5 orATTO 590)

1

1x***

Restriction enzyme,diluted*

1

2–5 U/reaction

DNA sample or water

Variable

50 fg–100 ng

Total Volume

20**–*

Table 4. Thermal cycling conditions.*†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.

5.

6.

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.

Cycling Step

Temperature, °C Time

Numberof Cycles

Hold (QX ONE ddPCRSystem only)

25

3 min

1

Enzyme activation95

10 min1

Denaturation

94

30 sec40

Annealing/extension

Optimum**

1 min***

40

Enzyme deactivation98

10 min1

Hold

4Infinite

1

QX ONE ddPCR 25System (required)

1 min

1

Transfer the reaction mix from the reaction tubes to theappropriate ddPCR Cartridge as follows:

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

– For the Automated Droplet Generator, follow
instructions in the Automated Droplet Generator
Instruction Manual (10043138)– 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 andQX ONE Software User Guide (10000116512)■■

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

QX200 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.
** Use optimal annealing temperature determined.
** Check/adjust ramp rate settings to ~2°C/sec– For the QX200 Droplet Generator, load 20 μl of eachreaction mix into a sample well of a DG8 Cartridge.
Follow subsequent instructions as specified in
the QX200 Droplet Generator Instruction Manual(10031907)■

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 for thisstep. Refer to the QX ONE Droplet Digital PCR System andQX ONE Software User Guide (10000116512) for plate setupinstructions. Use appropriate thermal cycling conditions as
specified in Table 4

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 4)

For the QX200 Droplet Reader, refer to the QX200 Droplet
Reader and QX Manager Software Standard Edition User
Guide (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 (10000116655
or 10000116656, respectively)

For the Automated Droplet Generator, remove the droplet
plate containing ddPCR droplets from the AutomatedDroplet Generator. Seal the plate using the PX1 PCR Plate
Sealer at 180°C for 5 sec. Proceed to thermal cycling (seeTable 4)© 2022 Bio-Rad Laboratories, Inc.

310000156273

ddPCR Mutation Detection Assays

1056304
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. 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 in additional Terms and Conditions, norights are granted for you to distribute or resell the Products. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted for the 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. and foreign patents and/orpending 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

10000156273 Ver A (10047489)

22-03590622