ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGCATTCTAGACATCTTCTAGATTCGTATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC ATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATT
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC ATTCTAGACATCTTCTAGATTCGTA
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGCATTCTAGACATCTTCTAGATTCGTAGTATATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCG
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC ATTCTAGACATCTTCTAGATTCG
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC
ATTCTAGACATCTTCTAGATTCGTAGTATGTCATAGCATTGTACCCGTCGTTACACATCTTCTAGATTCGTTACCATTCTAGATTCGC

Thermal Cycling

A Comparability Study of the C1000 Touch 96–Deep Well
and PTC Tempo Deepwell Thermal Cyclers for Use in
Droplet Digital™ PCR Workflow
Pallavi Shah, Oliver-Glenn Hernaez, Angelica Olcott, Candice Cox, Aki Uehara, Pengchin Chen, and Jimmie Lowery
Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547

Abstract

This study demonstrates the thermal performance between the PTC Tempo Deepwell Thermal Cycler
and the C1000 Touch Thermal Cycler with 96–Deep Well Reaction Module (C1000 Touch 96–Deep Well
Thermal Cycler) for amplification of input DNA prior to Droplet Dgital PCR (ddPCR™) applications on
the QX200™ and QX600™ Droplet Digital PCR Systems. The Bio-Rad QX600 ddPCR System delivers
best-in-class performance with the addition of six-color multiplexing. A previous study determinedthermal efficiency across a gradient, ramp rate, accuracy, and gradient performance, and demonstrated
comparable thermal performance of the PTC Tempo Deepwell and C1000 Touch 96–Deep Well Thermal
Cyclers (bulletin 3483). In this evaluation, the downstream amplification products provided by the
PTC Tempo Deepwell and C1000 Touch 96–Deep Well Thermal Cyclers were measured for their use
in a ddPCR workflow performed on the QX200 and QX600 ddPCR Systems, to determine the resulting
accuracy and precision of the two formats. This comparability study demonstrates that the accuracy
and precision of measurement of the DNA amplification products generated are comparable between
the PTC Tempo Deepwell and C1000 Touch 96–Deep Well Thermal Cyclers. The PTC Tempo Deepwell
Thermal Cycler is validated to support the Bio-Rad ddPCR workflow, specifically the QX200 and
QX600 Droplet Digital PCR Systems.
Introduction
For this evaluation of compatibility with Droplet Digital PCR, the
PTC Tempo Deepwell Thermal Cycler is used to amplify the DNA
target to endpoint within each droplet partition. After droplet
generation and PCR, each partition is read using a droplet reader(either with the QX200 or QX600 Droplet Reader) to determine thefraction of positive partitions that includes the target for calculationof target DNA concentration.
To ensure robust amplification, PTC Tempo Thermal Cyclers
were designed to include precise temperature control in a flexible
thermal gradient configuration. For DNA amplification in Droplet
Digital PCR, the PTC Tempo Deepwell Thermal Cycler is preferredas it accommodates larger volumes and provides a greater
amplification yield. Specifically, the deepwell reaction module offersa block height that ensures homogenous heating of the droplet
emulsion during Droplet Digital PCR.

Bulletin 3530

The PTC Tempo Deepwell Thermal Cycler builds on theperformance of its predecessor, the C1000 Touch 96–Deep Well
Thermal Cycler, and now includes user-friendly features such as an
automatic lid capable of autosensing plates and tubes, an LED light
display, audible status notifications, and connectivity options that
include the Bio-Rad BR.io cloud platform, WiFi, USB, and Ethernet
connections. Moreover, the PTC Tempo Deepwell Thermal Cycler
maintains the same user access controls for user managementwith the addition of enhanced security and reporting features.
This study evaluates the consistency in DNA concentration
measurements of samples obtained on the QX200 and QX600
ddPCR Systems using the PTC Tempo Deepwell Thermal Cycler
and the C1000 Touch 96–Deep Well Thermal Cycler. The study
shows its ability to provide high accuracy and precision of a DNA
target in ddPCR applications.

A Comparability Study of the C1000 Touch 96–Deep Well and PTC Tempo Deepwell Thermal Cyclers for Use in Droplet Digital PCR Workflow

Experimental Design
As shown in Figure 1, the experimental design was intended todemonstrate the comparability of amplified input DNA source
material for detection by Droplet Digital PCR on the QX200 and the
QX600 ddPCR Systems. The PTC Tempo Deepwell Thermal Cycler
was compared to the C1000 Touch 96–Deep Well Thermal Cycler.
Thermal Cycler

Table 1. Droplet Digital PCR assay metrics evaluated to compare amplified
input DNA input from the PTC Tempo Deepwell and C1000 Touch 96–DeepWell Thermal Cyclers.
Droplet Reader

Channels

QX600 Droplet
Reader

FAM, HEX,
Cy5, Cy5.5,
ROX, ATTO
590

Downstream ddPCR System

Metrics EvaluatednMean concentrationnAccuracy, compared to target concentrationnn

QX200 Droplet
Reader

FAM, HEXAverage droplet countnMean concentrationnAccuracy, compared to target concentrationn

C1000 Touch 96–Deep Well, n = 1

PCR-amplified
DNA source
materialn

QX200 Droplet Reader
Two detection channels

 ifference from control, PTC Tempo Deepwell vs.
D
C1000 Touch 96–Deep Well Thermal Cyclernn

PTC Tempo Deepwell, n = 3

 recision, % CV values for mean concentrationPin 6 detection channels

Precision, % CV values for mean concentrationin 2 detection channels
 ifference from control, PTC Tempo Deepwell vs.
D
C1000 Touch 96–Deep Well Thermal Cycler
Average droplet countCV, coefficient of variation.
Table 2. Thermal cycling protocol performed on the C1000 Touch 96–DeepWell and PTC Tempo Deepwell Thermal Cyclers.

QX600 Droplet Reader
Six detection channels

Step

Temperature

Time

Ramp

Number of cycles

1

95ºC

10 min2ºC/sec

1

2

94ºC

30 sec2ºC/sec

40

3

58ºC

1 min

2ºC/sec

40

4

98ºC

10 min2ºC/sec

1

5

4ºC

hold

2ºC/sec

1

Hold at 4ºC for at least 30 minutes prior to loading on QX200 or QX600 Droplet Reader.
Fig. 1. Comparability study experimental workflow for DNA amplification followed
by Droplet Digital PCR performed on QX600 and QX200 Droplet Readers.

QX600 Assay Procedure

The ddPCR Six-Color System Check Kit assay procedure uses a
proprietary validated reference DNA standard from the NMIA. This
NMIA reference DNA standard at 1,060 copies/µl was amplified on
both the PTC Tempo Deepwell and C1000 Touch 96–Deep Well
Thermal Cyclers, and the resulting amplified material was detectedon the QX600 Droplet Reader to determine accuracy, precision,and absolute concentration.

Materials and Methods
The ddPCR Six-Color System Check Kit* (Bio-Rad Laboratories, Inc.,
catalog #12016025) and QX200 Instrument Qualification Kit**(Bio-Rad, in development) assays were used in this study. Each
kit contains proprietary DNA templates that are manufactured
and quantitated by the National Measurement Institute of Australia(NMIA). Droplet generation was performed on the AutomatedDroplet Generator (Bio-Rad, #1864101) using DG32 AutomatedDroplet Generator Cartridges (Bio-Rad, #1864108), and AutomatedDroplet Generation Oil for Probes (Bio-Rad, #1864110). Detection
was performed on the QX200 Droplet Reader (Bio-Rad, #1864003)
and QX600 Droplet Reader (Bio-Rad, #12013328) using ddPCR
Droplet Reader Oil (Bio-Rad, #1863004) with input DNA obtained
from a total of four thermocyclers, three PTC Tempo Deepwell
Thermal Cyclers (Bio-Rad, #12015392) and one C1000 Touch
Thermal Cycler with 96–Deep Well Reaction Module (Bio-Rad,#1851197), respectively. The thermal cycler conditions used forproviding amplified DNA source material are listed in Table 2.

QX200 Assay Procedure

For the QX200 ddPCR System, quantitation of the proprietary
NMIA validated reference DNA standard at 1,060 copies/µl was
amplified using either a PTC Tempo Deepwell or C1000 Touch
96–Deep Well Thermal Cycler and then detected on the QX200
Droplet Reader to determine the accuracy, precision, andabsolute concentration.
Results
QX600 Droplet Reader Quantitation of NMIA Reference DNA Standard: Accuracy

The ddPCR Six-Color System Check assay was performed todetermine accuracy and precision using the NMIA DNA reference
standard on the C1000 Touch 96–Deep Well Thermal Cycler
and the PTC Tempo Deepwell Thermal Cyclers (Table 3). Results
exceeded all system specifications for minimum droplet count,variation from the NMIA standard reference concentration of 1,060
copies/µl, and concentration precision in each detection channel.

*	The ddPCR Six-Color System Check Kit is a Bio-Rad service kit to test system
performance to manufacturing specifications and is not available to order.
**	The QX200 Instrument Qualification Kit is in development and will be a Bio-Rad
service kit to test system performance to manufacturing specifications and is notavailable to order.

© 2023 Bio-Rad Laboratories, Inc.

2

Bulletin 3530

A Comparability Study of the C1000 Touch 96–Deep Well and PTC Tempo Deepwell Thermal Cyclers for Use in Droplet Digital PCR Workflow

Accurate quantification was provided by DNA reference standard
material and the ddPCR workflow with either thermal cycler
platform, with no deviations greater than ±1% from the NMIA
standard concentration (Table 3).

1,200

1,000

800
Copies/µl

Table 3. Comparable accuracy of reference DNA standard in the ddPCR
workflow with PTC Tempo Deepwell and C1000 Touch 96–Deep Well
Thermal Cyclers for the QX600 Droplet Reader. Both deep well thermal cyclers
provided similar performance. The concentration calls for the NMIA standard were
accurate on the QX600 Droplet Reader, with no deviations greater than ±1% from the
NMIA standard concentration.

400

PTC Tempo Deepwell
Metric200

C1000 Touch
96–Deep Well

Unit 1

Unit 2

Unit 3

1,056

1,053

1,050

1,052

Accuracy, detected
copies/μl,* NMIA target
1,060 copies/μl
Difference
fromcontrol**

—

–0.2%

–0.5%

0
C1000 Touch–0.4%* Detected copies/µl is calculated as the average concentration across a plate,detected by each channel (FAM, HEX, Cy5, Cy5.5, ROX, ATTO 590).
** C1000 Touch 96–Deep Well Thermal Cycler.

Results shown in Table 5 confirm that both thermal cycler formats
provided very similar uniform performance, based on the <1%
variation in concentration following Droplet Digital PCR on the
QX200 Droplet Reader.

Table 4. Precision of reference DNA standard quantitation in the ddPCR
workflow with PTC Tempo Deepwell and C1000 Touch 96–Deep Well
Thermal Cyclers using the QX600 Droplet Reader. Concentrations were
measured across all six detection channels (FAM, HEX, Cy5, Cy5.5, ROX, ATTO
590) with % CVs of <2.5%, and droplet counts were comparable for both deep wellthermal cycler formats across an entire plate.

Table 5. Comparable accuracy of amplified reference DNA standard fromPTC Tempo Deepwell and C1000 Touch 96–Deep Well Thermal Cyclers
for the QX200 Droplet Reader. Detection using the QX200 Droplet Reader isconsistent in the ddPCR workflow using either PTC Tempo Deepwell (three units)
or C1000 Touch 96–Deep Well (one unit) Thermal Cyclers in both FAM and HEX
detection channels and shows <1% variation in concentration.

PTC Tempo Deepwell

PTC Tempo Deepwell

C1000 Touch
96–Deep Well

Unit 1

Unit 2

Unit 3

FAM

2.0%

1.7%

2.3%

1.9%

HEX

2.2%

1.7%

2.4%

2.1%

Cy5

2.1%

1.6%

2.3%

2.1%

Difference fromcontrol**

Cy5.5

2.1%

1.7%

2.4%

1.9%

NMIA, National Measurement Institute of Australia.

ROX

2.1%

1.7%

2.3%

1.9%

2.2%

1.9%

2.4%

1.9%

21,426

21,280 20,332 20,452

ATTO 590
Droplet count, average
across a plate

MetricAccuracy, detected
copies/μl,* NMIA
target 1,060 copies/μl

C1000 Touch 96–Deep Well

Unit 1

Unit 2

Unit 3

1,024

1,030

1,029

1,029

—

+0.6%

+0.5%

+0.5%

* D
 etected copies/µl was calculated as average of concentration across a plate
detected by each channel (FAM, HEX).
** C1000 Touch 96–Deep Well Thermal Cycler.

QX200 Droplet Reader Quantitation of NMIA Reference DNA Standard:
Precision and Droplet CountCV, coefficient of variation.

As shown in Table 6, both ddPCR workflows using PTC Tempo
Deepwell and C1000 96–Touch Deep Well Thermal Cycler formats
provided very similar results for ddPCR precision with <2.4% CV
values for the mean concentration across a plate detected in the
FAM and HEX detection channels of the QX200 Droplet Reader.
Droplet counts are also similar when using either C1000 Touch
96–Deep Well Thermal Cycler or PTC Tempo Deepwell Thermal
Cycler for the ddPCR workflows.

QX600 Droplet Reader Quantitation of NMIA Reference DNA Standard:
Mean Concentration PlotAs shown in Figure 2, performance of the PTC Tempo Deepwell
and C1000 Touch 96–Deep Well Thermal Cyclers are comparable,
based on the consistent concentrations obtained following detectionof NMIA reference DNA standard on all six QX600 channels.
© 2023 Bio-Rad Laboratories, Inc.

PTC Tempo 3

The NMIA reference DNA standard at 1,060 copies/µl was
amplified by PTC Tempo Deepwell and C1000 Touch 96–DeepWell Thermal Cyclers and then detected on the QX200 Droplet
Reader to determine absolute concentration.

As shown in Table 4, the PTC Tempo Deepwell and the C1000
Touch 96–Deep Well Thermal Cycler formats provided very similar
results for ddPCR precision, with <2.5% CV values for the mean
concentration across a plate in all six detection channels of the
QX600 Droplet Reader. Droplet counts are also shown to be
similar using either the C1000 Touch 96–Deep Well or PTC Tempo
Thermal Cycler in the ddPCR workflow.

Precision, % CV,mean concentration

PTC Tempo 2

QX200 Droplet Reader Quantitation of NMIA Reference DNA Standard:
Accuracy and Droplet Count

QX600 Droplet Reader Quantitation of NMIA Reference DNA Standard:
Precision and Droplet Count

MetricPTC Tempo 1

Fig. 2. Comparable performance of PTC Tempo Deepwell and C1000 Touch
96–Deep Well Thermal Cyclers for mean concentration of reference DNA
standard across a plate. The ddPCR workflow with PTC Tempo Deepwell(three units) and C1000 Touch 96–Deep Well (one unit) Thermal Cyclers provided
consistent concentration calls across all six detection channels of the QX600 Droplet
Reader. NMIA standard target concentration of 1,060 copies/μl (), measured
average concentration across a plate FAM ( ); HEX ( ); Cy5 ( ); Cy5.5 ( ); ROX ( );ATTO 590 ( ). NMIA, National Measurement Institute of Australia.

NMIA, National Measurement Institute of Australia.

Detection
Channel

600

3Bulletin 3530

A Comparability Study of the C1000 Touch 96–Deep Well and PTC Tempo Deepwell Thermal Cyclers for Use in Droplet Digital PCR Workflow

Table 6. Precision of reference DNA standard DNA quantitation in the
ddPCR workflow with PTC Tempo Deepwell and C1000 Touch 96–Deep Well
Thermal Cyclers are similar using the QX200 Droplet Reader. <2.4% CV inconcentration was measured for two detection channels (FAM, HEX), and droplet
counts are comparable for both deep well thermal cycler formats. CV, coefficientof variation.
PTC Tempo Deepwell
Detection
ChannelMetricPrecision, % CV,mean concentration

C1000 Touch
96–Deep Well

Unit 1

Unit 2

Unit 3

FAM

2.3%

1.8%

1.8%

1.9%

HEX

2.1%

1.4%

1.6%

1.7%

18,822

19,407 19,359 19,739

Droplet count, average
across a plate

QX200 Quantitation of NMIA Reference DNA Standard: Mean Concentration PlotAs shown in Figure 3, PTC Tempo Deepwell and C1000 Touch
96–Deep Well Thermal Cycler performances are comparable,
based on the consistent concentrations obtained following thedetection of amplified input DNA on all QX200 Droplet Reader
channels.
1,200

1,000

Copies/ul

800

600

Conclusions
The Bio-Rad QX200 and QX600 Droplet Digital PCR Systems
provide precise and absolute quantification of nucleic acids,
and high quality thermal cycling is an important part of theddPCR worfklow.
This comparability study demonstrates that accurate and precise
nucleic acid measurements can be achieved with Droplet Digital
PCR using either the PTC Tempo Deepwell or the C1000 Touch
96–Deep Well Thermal Cycler and supports the adoption of the
PTC Tempo Deepwell Thermal Cycler by QX200 and QX600
Droplet Digital PCR System users.
Visit bio-rad.com/PTCTempo for more information.
BIO-RAD, DDPCR, DROPLET DIGITAL, QX200, and QX600 are trademarks of Bio-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 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,no rights are granted for you to distribute or resell the Products. Unless we expressly stateotherwise 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. It is the user’s obligation to obtain a commercial license from Bio-Rad for (i) allcommercial uses (not just diagnostic uses) and (ii) sale of assays for use on Bio-Rad’sdPCR and ddSEQ instruments. 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.

400

200

0
FAM HEX
C1000 Touch

FAM HEXPTC Tempo 1

FAM HEXPTC Tempo 2

FAM HEXPTC Tempo 3

Fig. 3. PTC Tempo Deepwell and C1000 Touch 96–Deep Well Thermal
Cycler performances are comparable for mean concentration of reference
DNA standard across a plate. The ddPCR workflow with the PTC Tempo
Deepwell and C1000 Touch 96–Deep Well Thermal Cyclers provided very uniform
concentration calls across all QX200 Droplet Reader detection channels, with similar
concentrations. NMIA standard target concentration of 1,060 copies/μl (),
measured average concentration across a plate FAM ( ); HEX ( ). NMIA, National
Measurement Institute of Australia.

Bio-RadLaboratories, Inc.
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