TaqPath™ ProAmp™ Master Mixes

USER GUIDE

Genotyping and copy number variation PCR workflows

Publication Number MAN0015758
Revision A.0

For Laboratory Use.

Manufacturer's address: Life Technologies Corporation | 2130 Woodward Street | Austin, TX 78744
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,
INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR
USE OF IT.

Revision history. Pub. No. MAN0015758

Revision Date Description
A.0 02 September 2016 New document.

Important Licensing Information. These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
Limited Use Label License No. 469: PCR General Purpose Reagent . Notice to Purchaser: For research purposes only. Diagnostic uses require a
separate license from Roche.
Limited Use Label License No. 475: General Purpose Reagents. Notice to Purchaser: The purchase of this product conveys to the purchaser the
limited, non-transferable right to use the purchased amount of the product only to perform internal research and development for the sole benefit of
the purchaser. No right to transfer or resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is
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additional rights, please contact [email protected] or Out Licensing, Life Technologies Corporation, 5781 Van Allen Way, Carlsbad, California
92008.
Trademarks. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered
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©2016 Thermo Fisher Scientific Inc. All rights reserved.
 Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ CHAPTER 2 Perform PCR for genotyping experiments . . . . . . . . . . . . . . . 8

Workflow: Genotyping experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Guidelines for input DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Guidelines for PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Set up the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Set up and run the real-time PCR instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ CHAPTER 3 Perform PCR for copy number experiments . . . . . . . . . . . 12

Workflow: Copy number experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Guidelines for input DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Guidelines for PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Set up the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Set up and run the real-time PCR instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

TaqPath™ ProAmp™ Master Mixes User Guide 3

Contents

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ APPENDIX B Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Master mix components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
™
Dual-Lock Taq DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Heat-labile uracil-N-glycosylase (UNG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
dUTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
™
MUSTANG PURPLE dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Best practices for PCR and RT-PCR experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Use UNG to prevent false-positive amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Detect fluorescent contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

4 TaqPath™ ProAmp™ Master Mixes User Guide

 1 Product information

Product description
Applied Biosystems™ TaqPath™ ProAmp™ Master Mixes are optimized for genotyping
and copy number (CNV) experiments that use samples extracted from various human
or animal sources, including blood cards, buccal swabs, and whole blood.
TaqPath™ ProAmp™ Master Mixes are formulated for:
• Extended shelf life and benchtop stability
• Enhanced specificity and inhibitor tolerance during PCR for:
– Samples with stubborn inhibitors
– Samples from crude lysates (with most genotyping assays)
Note: For copy number assays, crude lysates are not recommended because
the input DNA concentration and quality cannot be determined accurately.
See Table 4 for compatible genotyping and copy number TaqMan® Assays.
TaqPath™ ProAmp™ Master Mixes are supplied at a 2X concentration and contain:
• Dual-Lock™ Taq DNA Polymerase
• Heat-labile uracil-DNA glycosylase (UNG)
• dNTPs with dUTP
• Passive reference dye (ROX™ –or– MUSTANG PURPLE™)
• Optimized buffer components
For more information about the components of TaqPath™ ProAmp™ Master Mixes, see
“Master mix components“ on page 17.

Table 1 Recommended master mix formulation for single- and multiplex reactions

Compatible
PCR option[1] Recommended master mix formulation
reporter dyes

Singleplex (1 probe)
FAM™, VIC™, ABY™ TaqPath™ ProAmp™ Master Mix with ROX™
Multiplex (2–3 probes)

TaqPath™ ProAmp™ Multiplex Master Mix with MUSTANG PURPLE™

JUN™ plus Note: See your instrument user guide about additional calibration that
Multiplex (≥ 3 probes)
FAM™, VIC™, ABY™ may be required for use of MUSTANG PURPLE™ dye, including any
additional spectral calibration plates that may be sold separately.
[1] For detailed information about multiplex reactions, see the TaqMan® Multiplex PCR Optimization User Guide (Pub. No. MAN0010189).

TaqPath™ ProAmp™ Master Mixes User Guide 5

1 Chapter 1 Product information
Contents and storage

Contents and storage

Table 2 TaqPath™ ProAmp™ Master Mix formulations

TaqPath™ ProAmp™ TaqPath™ ProAmp™ Multiplex

Master Mix Master Mix Amount Storage
™ ™
with ROX with MUSTANG PURPLE

A30865 A30868 1 × 1 mL

A30866 A30869 1 × 10 mL

A30871 A30873 2 × 10 mL 2–8°C

A30867 A30870 1 × 50 mL

A30872 A30874 2 × 50 mL

Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com.
MLS: Fisher Scientific (www.fisherscientific.com) or other major laboratory supplier.

Table 3 Recommended DNA isolation kits

Kit Cat. No.

A25597
MagMAX™ DNA Multi-Sample Ultra Kit
A25598

DNA Extract All Reagents Kit 4402599

Table 4 Compatible TaqMan® Assays

Assay collection Source

TaqMan® SNP Genotyping Assays thermofisher.com/taqmansnp

TaqMan® Drug Metabolism Genotyping Assays thermofisher.com/taqmandme

TaqMan® Copy Number Assays thermofisher.com/taqmancnv

6 TaqPath™ ProAmp™ Master Mixes User Guide

 Chapter 1 Product information
Required materials not supplied 1

Table 5 Other materials and equipment required for PCR reactions

Item Source

Real-time PCR instrument, one of the following:

QuantStudio™ 3 or 5 Real-Time PCR System

QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System

QuantStudio™ 12K Flex Real-Time PCR System

QuantStudio™ Dx Real-Time PCR Instrument Contact your local

™
ViiA 7 Real-Time PCR System sales office

ViiA™ 7 Dx Real‑Time PCR Instrument

7500 Fast Real-Time PCR System

StepOne™ or StepOnePlus™ Real-Time PCR System[1,2]

Equipment

Centrifuge with plate adapter MLS

Microcentrifuge MLS

Single- or multi-channel pipettes (electronic or manual) MLS

Laboratory mixer (vortex or equivalent) MLS

Tubes, plates, and other consumables

thermofisher.com/
Plastics consumables
plastics

Aerosol-resistant barrier pipette tips MLS

Disposable gloves MLS

[1] The StepOne and StepOnePlus Systems are not compatible with TaqPath ProAmp Multiplex Master Mix.
™ ™ ™ ™

[2] The StepOne™ Real-Time PCR System is not compatible with the requirements for copy number experiments.

TaqPath™ ProAmp™ Master Mixes User Guide 7

 2 Perform PCR for genotyping
experiments

Workflow: Genotyping experiments

“Set up the PCR reactions“ on page 9

“Set up and run the real-time PCR instrument“ on page 10

“Analyze the results“ on page 11

Procedural guidelines
Guidelines for • Use a compatible DNA isolation kit (see Table 3 on page 6).
input DNA • All wells that use the same assay must contain similar amounts of sample DNA.
• For genotyping experiments, use a final DNA concentration of at least 0.2 ng/µL
for each reaction.
• For crude lysates, sample volume can be up to 25% of the total reaction volume.
Use the DNA Extract All Reagents Kit (Cat. No. 4402599) for crude lysate
preparation.

Guidelines for • For reaction volumes that are different from those detailed, scale all components
PCR reactions proportionally and include 10% overage.
• For genotyping experiments, no template control (NTC) reactions are required to
call the genotype. NTC reactions can also be used to identify PCR contamination.
NTC reactions contain all reaction components (master mix, assays, water) except
DNA sample.

8 TaqPath™ ProAmp™ Master Mixes User Guide

 Chapter 2 Perform PCR for genotyping experiments
Set up the PCR reactions 2

Set up the PCR reactions

1. Prepare the required number of reactions according to the following table, plus
10% overage.

Table 6 PCR reactions for genotyping experiments

Volume per reaction

Component 5 µL 10 µL 25 µL
(384-well) (96-well Fast) (96-well Std)
TaqPath™ ProAmp™ Master Mix 2.5 µL 5.0 µL 12.5 µL
TaqMan® SNP Genotyping Assay[1]
0.25 µL 0.5 µL 1.25 µL
(20X)
Genomic DNA -or- NTC [2] up to 2.25 µL up to 4.5 µL up to 11.25 µL
Nuclease-Free Water to 5 µL total to 10 µL total to 25 µL total
Total volume 5 µL 10 µL 25 µL
[1] For Research Use Only. Not for use in diagnostic procedures.
[2] For crude lysates, sample volume can be up to 25% of the total reaction volume.

2. Mix the components thoroughly, then centrifuge briefly to spin down the
contents and eliminate air bubbles.

3. Add the appropriate volume of each reaction to each well of an optical plate.

4. Seal the plate with an optical adhesive cover, then centrifuge briefly to spin down
the contents and eliminate air bubbles.

(Optional) Store the reaction plate for up to 72 hours at room temperature.

TaqPath™ ProAmp™ Master Mixes User Guide 9

2 Chapter 2 Perform PCR for genotyping experiments
Set up and run the real-time PCR instrument

Set up and run the real-time PCR instrument

1. Load the reaction plate in the real-time PCR instrument.

2. Set the appropriate experiment settings and PCR thermal cycling conditions.
Select genotyping as the experiment type, and select the correct passive reference
for the master mix formulation.
Note: Heat-labile UNG is active during the reaction setup and is completely
inactivated during the first ramp to the 95°C hold step.
Note: Fast cycling is not recommended for TaqMan® Drug Metabolism
Genotyping Assays.

Table 7 Genotyping experiments: standard cycling

Step Temperature Time Cycles

Pre-Read 60°C 30 seconds
Initial denature / Hold
95°C 5 minutes
Enzyme activation
Denature 95°C 15 seconds
40
Anneal / Extend 60°C 60 seconds[1]
Post-Read 60°C 30 seconds Hold
[1] For Drug Metabolizing Enzyme (DME) assays, set duration to 90 seconds.

Table 8 Genotyping experiments: fast cycling

Step Temperature Time[1] Cycles

Pre-Read 60°C 30 seconds
Initial denature / Hold
95°C 5 minutes
Enzyme activation
Denature 95°C 5 seconds
40
Anneal / Extend 60°C 30 seconds
Post-Read 60°C 30 seconds Hold
[1] Optional cycling for Fast 96-well or 384-well plates only.

3. Set the reaction volume appropriate for the reaction plate.

4. Start the run.

(Optional) After PCR, store the reaction plate protected from light:
• Up to 24 hours at room temperature.
• Up to 72 hours at 4°C.

10 TaqPath™ ProAmp™ Master Mixes User Guide

 Chapter 2 Perform PCR for genotyping experiments
Analyze the results 2

Analyze the results

For detailed information about data analysis, see the appropriate documentation for
your assay and real-time PCR instrument.
Analyze allelic discrimination plots for genotyping data using any of the following
tools:

Software Resource

Thermo Fisher Cloud

thermofisher.com/us/en/home/cloud.html
GT Analysis module

TaqMan® Genotyper Software Getting Started Guide

TaqMan® Genotyper software
(Pub. No. 4448637)

Real-time PCR system

See “Related documentation“ on page 22
analysis software

For general information about SNP genotyping experiments and analysis, see the
TaqMan® SNP Genotyping Assays User Guide (Pub. No. MAN0009593).

TaqPath™ ProAmp™ Master Mixes User Guide 11

 3 Perform PCR for copy number
experiments

Workflow: Copy number experiments

“Set up the PCR reactions“ on page 13

“Set up and run the real-time PCR instrument“ on page 14

“Analyze the results“ on page 14

Procedural guidelines
Guidelines for • Use a compatible DNA isolation kit (see Table 3 on page 6).
input DNA • Use purified gDNA as the DNA input; we do not recommend using crude lysate
for copy number (CNV) experiments.
• All wells that use the same assay must contain similar amounts of sample DNA.
• For copy number experiments, use a final purified DNA concentration of 1 ng/µL
for each reaction.

Guidelines for • For copy number experiments, we recommend four replicates of each reaction.
PCR reactions • For reaction volumes that are different from those detailed, scale all components
proportionally and include 10% overage.
• NTC reactions can be used to identify PCR contamination.
NTC reactions contain all reaction components (master mix, assays, water) except
DNA sample. These reactions should not result in amplification.

12 TaqPath™ ProAmp™ Master Mixes User Guide

 Chapter 3 Perform PCR for copy number experiments
Set up the PCR reactions 3

Set up the PCR reactions

1. Prepare the required number of reactions according to the following table, plus
10% overage.

Table 9 PCR reactions for copy number experiments

Volume per reaction

Component 10 µL 20 µL
(384-well) (96-well Std)
TaqPath™ ProAmp™ Master Mix 5.0 µL 10.0 µL
TaqMan® Copy Number Assay[1]
0.5 µL 1.0 µL
(20X)
TaqMan® Copy Number Reference
0.5 µL 1.0 µL
Assay[1] (20X)
Genomic DNA -or- NTC up to 4 µL up to 8 µL
Nuclease-Free Water to 10 µL total to 20 µL total
Total volume 10 µL 20 µL
[1] For Research Use Only. Not for use in diagnostic procedures.

2. Mix the components thoroughly, then centrifuge briefly to spin down the
contents and eliminate air bubbles.

3. Add the appropriate volume of each reaction to each well of an optical plate.

4. Seal the plate with an optical adhesive cover, then centrifuge briefly to spin down
the contents and eliminate air bubbles.

(Optional) Store the reaction plate for up to 72 hours at room temperature.

TaqPath™ ProAmp™ Master Mixes User Guide 13

3 Chapter 3 Perform PCR for copy number experiments
Set up and run the real-time PCR instrument

Set up and run the real-time PCR instrument

1. Load the reaction plate in the real-time PCR instrument.

2. Set the appropriate experiment settings and PCR thermal cycling conditions.
Select standard curve as the experiment type, and select the correct passive
reference for the master mix formulation.
Note: Heat-labile UNG is active during the reaction setup and is completely
inactivated during the first ramp to the 95°C hold step.

Table 10 Copy number experiments: standard cycling

Step Temperature Time Cycles

Initial denature /
95°C 10 minutes Hold
Enzyme activation
Denature 95°C 15 seconds
40
Anneal / Extend 60°C 60 seconds

3. Set the reaction volume appropriate for the reaction plate.

4. Start the run.

Analyze the results

For detailed information about data analysis, see the appropriate documentation for
your assay and real-time PCR instrument.
We recommend using the following options in the analysis settings:
• Select the auto-baseline setting.
• Manually set the threshold values.
For copy number experiments, set the thresholds for the reference assay and the
copy number target assay to the same value.

Recommended
Master mix formulation
threshold value
TaqPath™ ProAmp™ Master Mix 0.2
TaqPath™ ProAmp™ Multiplex Master Mix 0.8

Analyze copy number data using CopyCaller™ Software; see the CopyCaller™
Software v2.0 User Guide (Pub. No. 4400042).
For general information about copy number experiments and analysis, see the
TaqMan® Copy Number Assays Protocol (Pub. No. 4397425).

14 TaqPath™ ProAmp™ Master Mixes User Guide

 A Troubleshooting

Observation Possible cause Recommended action

Small particles during reaction mix setup The particles are components No action is required. The
of the master mix. particles do not interfere with
the reaction and do not affect
physical apparatus, including
robotics.
Amplification plot shows a high level of noise The incorrect dye is set as the Change the passive reference
passive reference in the run to the correct dye, then
file. reanalyze the data.
• TaqPath™ ProAmp™
Master Mix—ROX™
• TaqPath™ ProAmp™
Multiplex Master Mix—
MUSTANG PURPLE™

Unexpected results for genotyping experiments The assay that was used for Use a purified DNA sample
using crude lysates the genotyping experiment is input for use with the chosen
not compatible with crude genotyping assay.
lysates.
High NTC signal for genotyping experiments Some genotyping assays have No action is required. The
a significant NTC signal that signal does not contribute to
is not due to amplification; or interfere with the genotype
rather, it is a property of the cluster signal or analysis.
assay. In the absence of DNA
template, some dye cleavage
may occur.

TaqPath™ ProAmp™ Master Mixes User Guide 15

A Appendix A Troubleshooting
Analyze the results

Observation Possible cause Recommended action

Amplification curves for samples appear as When automatic baseline is For copy number
sigmoidal curves used, the software selects experiments: When analyzing
baseline cycle values that are the data, change the analysis
too narrow. setting to allow manual
adjustment, then set the
Baseline Start Cycle to 3 and
the Baseline End Cycle to 15.
Set the Amplification Plot's
graph type to linear. If
amplification appears to
begin earlier than cycle 15,
adjust the Baseline End Cycle
to 2 cycles prior to the start of
amplification. For example, if
the amplification appears to
begin at cycle 13, set the Start
Cycle to 3 and End Cycle to
11.
The Ct is low When automatic baseline is For copy number
used, the software selects experiments: When analyzing
Note: Related observations are false positives or
baseline cycle values that are the data, change the analysis
high NTC signal.
too narrow. setting to allow manual
adjustment, then set the
Baseline Start Cycle to 3 and
the Baseline End Cycle to 15.
Evaluate the multi-
component plot to ensure that
the Ct signal represents a
true amplification and not
part of the background signal
noise.

16 TaqPath™ ProAmp™ Master Mixes User Guide

 B Supplemental information

Master mix components

Dual-Lock™ Taq This master mix contains Dual-Lock™ Taq DNA Polymerase that uses a combination
DNA Polymerase of two hot-start mechanisms to control its activity. By providing tight control over Taq
activation, this dual hot-start approach helps to prevent undesirable early activity of
the polymerase at low temperatures that can lead to nonspecific amplification.
The polymerase is provided in an inactive state to automate the hot-start PCR and
allow flexibility in the reaction setup, including pre-mixing of PCR reagents at room
temperature. The polymerase is activated after a brief hold step at 95°C.

Heat-labile uracil- This master mix contains heat-labile uracil-N-glycosylase (UNG; also known as
N-glycosylase uracil-DNA glycosylase (UDG)). Heat-labile UNG prevents reamplification of
carryover PCR products.
(UNG)
Treatment with UNG degrades dU-containing PCR carryover products and
misprimed, nonspecific DNA molecules. UNG acts on single- and double-stranded
dU-containing DNA by hydrolyzing uracil-glycosidic bonds at dU-containing DNA
sites. The enzyme causes the release of uracil and creates an alkali-sensitive
apyrimidic site in the DNA. Apyrimidic sites block replication by DNA polymerases.
The enzyme has no activity on RNA or dT-containing DNA.
UNG enzymatic activity occurs during the PCR reaction setup at room temperature;
an activation step before thermal cycling is not necessary. Unlike standard UNG,
heat-labile UNG is completely inactivated during the first ramp to the high-
temperature step for template denaturation and polymerase activation.
PCR products from reactions that include heat-labile UNG are stable for up to
72 hours post-amplification.

dUTP This master mix includes dUTP to enable uracil-N-glycosylase (UNG) activity and
maintain optimal PCR results.

MUSTANG MUSTANG PURPLE™ dye can act as a passive reference (absorption 647 nm;
PURPLE™ dye emission 654 nm) to provide an internal reference for normalizing the reporter-dye
signal during data analysis for multiplex reactions. Normalization corrects for
fluorescence fluctuations due to changes in concentration or volume.
For multiplex reactions, MUSTANG PURPLE™ dye can be used instead of the typical
ROX™ reference dye. This replacement allows the use of JUN™ dye which is detected
by the same channel as ROX™ dye.
For more information about multiplex reactions, see the TaqMan® Multiplex PCR
Optimization User Guide (Pub. No. MAN0010189).

TaqPath™ ProAmp™ Master Mixes User Guide 17

B Appendix B Supplemental information
Best practices for PCR and RT-PCR experiments

Best practices for PCR and RT-PCR experiments

Good laboratory When preparing samples for PCR or RT-PCR amplification:
practices for PCR • Wear clean gloves and a clean lab coat (not previously worn while handling
and RT-PCR amplified products or during sample preparation).
• Change gloves whenever you suspect that they are contaminated.
• Maintain separate areas and dedicated equipment and supplies for:
– Sample preparation and reaction setup.
– Amplification and analysis of products.
• Do not bring amplified products into the reaction setup area.
• Open and close all sample tubes carefully. Avoid splashing or spraying samples.
• Keep reactions and components capped as much as possible.
• Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips.
• Clean lab benches and equipment periodically with 10% bleach solution or
DNAZap™ Solutions (Cat. No. AM9890).

Use UNG to Carryover amplicons can result in false-positive amplification during PCR. Use a
prevent false- master mix that contains heat-labile uracil-N-glycosylase (UNG; also known as
uracil-DNA glycosylase (UDG)) to degrade many contaminating carryover amplicons.
positive
amplification UNG enzymatic activity occurs during the PCR reaction setup at room temperature;
an activation step before thermal cycling is not necessary. Unlike standard UNG,
heat-labile UNG is completely inactivated during the first ramp to the high-
temperature step for template denaturation and polymerase activation.
To ensure the desired UNG activity:
• Use PCR components and thermal cycling conditions as specified.
UNG-containing master mixes incorporate the optimal concentration of UNG to
prevent cross-contamination while not affecting real-time PCR performance.
• Do not attempt to use UNG-containing master mixes in subsequent amplification
of dU-containing PCR products, such as in nested-PCR protocols. The UNG will
degrade the dU-containing PCR products, preventing further amplification.
Although treatment with UNG can degrade or eliminate large numbers of carryover
PCR products, use good laboratory practices to minimize cross-contamination from
non-dU-containing PCR products or other samples.

Detect fluorescent Fluorescent contaminants can generate false positive results. To help detect these
contaminants contaminants, we recommend including a No-Amplification Control reaction that
contains sample, but no master mix.
After PCR, if the absolute fluorescence of the No-Amplification Control is greater than
the fluorescence of the no template control (NTC), fluorescent contaminants may be
present in the sample or in the heat block of the real-time PCR instrument.

18 TaqPath™ ProAmp™ Master Mixes User Guide

 C Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified

in the user documentation may result in personal injury or damage to the
instrument or device. Ensure that anyone using this product has received
instructions in general safety practices for laboratories and the safety
information provided in this document.
· Before using an instrument or device, read and understand the safety
information provided in the user documentation provided by the
manufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety Data
Sheets (SDSs) and use appropriate personal protective equipment (gloves,
gowns, eye protection, etc). To obtain SDSs, see the “Documentation and
Support” section in this document.

TaqPath™ ProAmp™ Master Mixes User Guide 19

C Appendix C Safety
Chemical safety

Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,
ensure laboratory personnel read and practice the general safety guidelines for
chemical usage, storage, and waste provided below. Consult the relevant SDS
for specific precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by the
chemical manufacturer before you store, handle, or work with any chemicals
or hazardous materials. To obtain SDSs, see the “Documentation and
Support” section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood).
· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow
the manufacturer's cleanup procedures as recommended in the SDS.
· Handle chemical wastes in a fume hood.
· Ensure use of primary and secondary waste containers. (A primary waste
container holds the immediate waste. A secondary container contains spills
or leaks from the primary container. Both containers must be compatible
with the waste material and meet federal, state, and local requirements for
container storage.)
· After emptying a waste container, seal it with the cap provided.
· Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
· Ensure that the waste is stored, transferred, transported, and disposed of
according to all local, state/provincial, and/or national regulations.
· IMPORTANT! Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.

20 TaqPath™ ProAmp™ Master Mixes User Guide

 Appendix C Safety
Biological hazard safety C

Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,
infectious agents, and blood of humans and other animals have the potential to
transmit infectious diseases. Conduct all work in properly equipped facilities
with the appropriate safety equipment (for example, physical containment
devices). Safety equipment can also include items for personal protection, such
as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety
glasses, or goggles. Individuals should be trained according to applicable
regulatory and company/ institution requirements before working with
potentially biohazardous materials. Follow all applicable local, state/provincial,
and/or national regulations. The following references provide general
guidelines when handling biological samples in laboratory environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiological
and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)
21-1112, Revised December 2009; found at:
www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf
· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

TaqPath™ ProAmp™ Master Mixes User Guide 21

 Documentation and support

Related documentation
Document Pub. No.

TaqPath™ ProAmp™ Master Mixes Quick Reference 100040972

TaqMan® SNP Genotyping Assays User Guide MAN0009593

TaqMan® Copy Number Assays Protocol 4397425

Instrument documentation

QuantStudio™ Design and Analysis desktop Software User Guide MAN0010408

QuantStudio™ 6 and 7 Flex Real-Time PCR System Software Getting Started Guide 4489822

QuantStudio™ 12K Flex Real-Time PCR System: Multi‐Well Plates and Array Card
4470050
Experiments User Guide

Applied Biosystems™ ViiA™ 7 Real-Time PCR System Getting Started Guide 4441434

Applied Biosystems™ 7500/7500 Fast Real‐Time PCR System Getting Started Guide:
4387784
Genotyping Experiments

Applied Biosystems™ 7500/7500 Fast Real‐Time PCR System Getting Started Guide:
4387779
Standard Curve Experiments

Applied Biosystems™ StepOne™ and StepOnePlus™ Real-Time PCR Systems

4376786
Genotyping Experiments Getting Started Guide

Data analysis

See thermofisher.com/us/
Thermo Fisher Cloud GT Analysis module
en/home/cloud.html

TaqMan® Genotyper Software Getting Started Guide 4448637

TaqMan® SNP Genotyping Assays User Guide MAN0009593

CopyCaller™ Software v2.0 User Guide 4400042

22 TaqPath™ ProAmp™ Master Mixes User Guide

 Documentation and support
Customer and technical support

Customer and technical support

Visit thermofisher.com/support for the latest in services and support, including:
• Worldwide contact telephone numbers
• Product support, including:
– Product FAQs
– Software, patches, and updates
– Training for many applications and instruments
• Order and web support
• Product documentation, including:
– User guides, manuals, and protocols
– Certificates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers,
contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth
in the Life Technologies' General Terms and Conditions of Sale found on Life
Technologies' website at www.thermofisher.com/us/en/home/global/
terms-and-conditions.html. If you have any questions, please contact Life
Technologies at www.thermofisher.com/support.

TaqPath™ ProAmp™ Master Mixes User Guide 23

For support visit thermofisher.com/support or email [email protected]
thermofisher.com

2 September 2016