A guide to real-time PCR reagents and consumables

Reagents and Consumables

Sequence Detection Systems
Introduction
Applied Biosystems Sequence Detection Systems (SDS) provide Default Primer and Probe Concentrations
everything you need to perform real-time PCR using either the fluoro- Applied Biosystems has developed a range of Master Mix configurations
genic 5' nuclease assay or SYBR Green 1 double-stranded DNA designed specifically to work within the Rapid Assay Development
binding dye chemistry. The complete system includes instrumenta- Guidelines. When using these Master Mix configurations with DNA or
tion, application specific software, reagents, consumables, and cDNA as the substrate, primer (and probe) optimization can be
protocols. Each component of the system is designed and optimized eliminated in favor of default concentrations.
to work together, ensuring a rapid and efficient path to REACTION FORWARD PRIMER REVERSE PRIMER PROBE
TYPE CONCENTRATION CONCENTRATION CONCENTRATION
experimental results. (nM) (nM) (nM)
DNA PCR 900* 900* 250
Rapid Assay Development Guidelines using TaqMan
Universal PCR
Applied Biosystems has developed a comprehensive set of guidelines to Master Mix
ensure success when using Sequence Detection Systems reagents and
instrumentation. These guidelines are simple and easy to follow. Many DNA PCR using 50 50 N/A
traditional variables, such as magnesium chloride concentration and the SYBR Green
thermal cycling protocol itself, have been standardized, greatly reducing PCR Master Mix
assay development time. *When performing a multiplex reaction with two reporter dyes, it is necessary to
limit the concentrations of the primers amplifying the more abundant target.
The Rapid Assay Development Guidelines consist of the following steps:
Primer and probe design using Primer Express software

Internal Passive Reference
Selecting the appropriate reagent (see table below) All SDS reagent kits contain a passive internal reference to normalize
Universal thermal cycling parameters non-PCR related fluorescence fluctuations. Normalizing with a passive
Assay optimization internal reference minimizes well-to-well variability that can result from
a variety of causes, such as pipetting errors and sample evaporation. The
passive reference is also essential for accurate results when multiple
probes (with different reporter dyes) are combined in a single tube.

Recommended Reagent Configurations by Application

Application Reaction Type Recommended TaqMan Recommended SYBR
Assay Reagent Green 1 Assay Reagent
RNA Quantitation One-Step RT-PCR TaqMan One-Step RT-PCR Master SYBR Green RT-PCR Reagents
Mix Reagents Kit (P/N 4309169)* (P/N 4310179)
TaqMan EZ RT-PCR Core Reagents

(when a high-temperature RT step

is required) (P/N N808-0236)*
Two-Step RT-PCR RT Step TaqMan Reverse Transcription RT Step TaqMan Reverse
Reagents (P/N N808-0234) Transcription Reagents
(P/N N808-0234)
PCR Step TaqMan Universal PCR PCR Step SYBR Green PCR

Master Mix (P/N 4304437) Master Mix (when AmpErase UNG

is not required) (P/N 4309155)
SYBR Green PCR Core Reagents

(when AmpErase UNG is required)

DNA Quantitation
(P/N 4304886)
DNA PCR TaqMan Universal PCR Master Mix SYBR Green PCR Master Mix (when
(P/N 4304437) AmpErase UNG is not required)
(P/N 4309155)
SYBR Green PCR Core Reagents

(when AmpErase UNG is required)

(P/N 4304886)*
SNP Genotyping (Allelic DNA PCR TaqMan Universal PCR Master Mix Not Applicable
Discrimination Assays) (P/N 4304437)
Plus/Minus Assays DNA PCR TaqMan Exogenous Internal Positive Not Applicable
using an IPC (Internal Control Reagents with TaqMan Universal
Positive Control) PCR Master Mix (P/N 4308320)
TaqMan PCR kits
TaqMan Universal PCR Master Mix*
TaqMan Universal PCR Master Mix combines components of the The fluorogenic 5' nuclease assay is a convenient, self-contained
TaqMan PCR Core Reagents Kit with additional key components in one process. The assay uses a fluorogenic probe consisting of an
easy-to-use premix. Proprietary buffer components and stabilizers are oligonucleotide to which a reporter dye and a quencher dye are
optimized to enhance reaction performance across all sample types, even attached. During PCR, the probe anneals to the target of interest
when the most demanding G/C rich templates are encountered. Enzyme
between the forward and reverse primer sites. During extension, the
concentration is also optimized to give superior performance in multiplex
probe is cleaved by the 5' nuclease activity of the DNA polymerase.
assay systems. Input sample template should be DNA or cDNA.
This separates the reporter dye from the quencher dye, generating an
Recommended storage conditions are 2-8C, so no thawing is required
before use. increase in the reporter dyes fluorescence intensity. (See diagram.)
DESCRIPTION QUANTITY PART NO.
TaqMan Universal PCR Master Mix 200 reactions 4304437
1 bottle, 5 mL
2X Solution Contains
AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs (with dUTP), POLYMERIZATION
Passive Reference 1, and optimized buffer components (proprietary formulation)
FORWARD
TaqMan Universal PCR 4304449 PRIMER R P ROBE Q
5' 3'
Master Mix Protocol 3' 5'
10-pack, TaqMan Universal PCR 2000 reactions 4305719 5' 3'
5'
Master Mix REVERSE
PRIMER
TaqMan Core Reagents*
The reporter (R) and the quencher (Q) dyes are attached to the probe.
DESCRIPTION QUANTITY PART NO.

TaqMan PCR Core Reagents Kit 200 reactions N808-0228
Contains
AmpliTaq Gold DNA Polymerase, 5 U/L, 50 L, 250 units total STRAND DISPLACEMENT R
Q
10X TaqMan Buffer A, 1.2 mL, 500 mM KCl, 100 mM Tris-HCl, 0.1 mM EDTA, 5' 3'
Passive Reference 1, pH 8.3 at room temperature (solution has been autoclaved) 3' 5'
AmpErase UNG, 1 U/L, 100 L, 100 units total 5' 3'
dATP, dCTP, and dGTP, 320 L, 10 mM each 5'
dUTP, 320 L, 20 mM
MgCl2 solution, 2 tubes, 1.5 mL each, 25 mM When both dyes are attached to the probe, reporter dye emission
TaqMan PCR Core Reagents Kit Protocol 402823 is quenched.
10-pack, TaqMan PCR 2000 reactions 402930
Core Reagents Kit
CLEAVAGE R
TaqMan PCR Core Reagents 200 reactions N808-0230 Q
5' 3'
Kit with Controls 3' 5'
Contains 5' 3'
TaqMan PCR Core Reagents Kit (N808-0228) 5'
TaqMan -Actin Detection Reagents (401846)
Protocol (402823) During extension, DNA polymerase cleaves the reporter dye from
TaqMan 1000 Reactions PCR 1000 reactions 4304439 the probe.
Core Reagents Kit
Contains Q
AmpliTaq Gold DNA Polymerase, 5 U/L, 250 L, 1250 units total POLYMERIZATION COMPLETED R 3'
10X TaqMan Buffer A, 6 mL, 500 mM KCl, 100 mM Tris-HCl, 0.1 mM EDTA, 5'
Passive Reference 1, pH 8.3 at room temp (solution has been autoclaved) 3' 5'
AmpErase UNG, 1U/L, 0.5 mL, 500 units total 5' 3'
dATP, dCTP, dGTP, 1.6 mL, 10 mM each 5'
dUTP, 1.6 mL, 20 mM
MgCl2 solution, 15 mL, 25 mM Once separated from the quencher, the reporter dye emits its characteristic
TaqMan 1000 RXN Gold with 1000 reactions 4304441 fluorescence. The ABI PRISM 7700 and GeneAmp 5700 Sequence
Buffer A Pack Detection Systems measure the increase in the reporter dyes fluorescence
Contains
AmpliTaq Gold DNA Polymerase, 5 U/L, 250 L, 1250 units total during the thermal cycling of the PCR. This data is then used by the
10X TaqMan Buffer A, 6 mL, 500 mM KCl,100 mM Tris-HCl, 0.1 mM EDTA, sequence detection software to generate quantitative results.
Passive Reference 1, pH 8.3 at room temp (solution has been autoclaved)
MgCl2 solution, 15 mL, 25 mM

*Purchase of this product is accompanied by a limited license under U.S. Patent 5,035,996 and foreign equivalents to use for research.
 TaqMan
RT-PCR kits
TaqMan One-Step RT-PCR Master Mix Reagents* TaqMan EZ RT-PCR Core Reagents 402877
The TaqMan One-Step RT-PCR Master Mix Reagents combine many of Protocol
the key components included in TaqMan Universal PCR Master Mix into 10-pack, TaqMan EZ RT-PCR 2000 reactions 403028
a master mix designed for one-step RT-PCR. The result is an easy to use Core Reagents
system providing excellent performance even when the most demanding
G/C rich templates are encountered. Enzyme concentrations are also TaqMan EZ RT-PCR Core Reagents 200 reactions N808-0235
optimized to give superior performance in multiplex assay systems. The With Controls
Contains
kit contains two components with recommended storage conditions of TaqMan EZ RT-PCR Core Reagents (N808-0236)
2-8C, so no thawing is required before use. TaqMan Human GAPDH Control Reagents (402869)
DESCRIPTION QUANTITY PART NO. Protocol (402877)
TaqMan One-Step RT-PCR 200 Reactions 4309169
TaqMan Gold RT-PCR Kit
Master Mix Reagents
Contains
The TaqMan Gold RT-PCR Kit provides all the necessary components to
2X Master Mix (5.0 mL) which includes AmpliTaq Gold DNA Polymerse, dNTPs (with perform both one and two-step RT-PCR, ideal for evaluation between the
dUTP), Passive Reference 1, and optimized buffer components (proprietary formulation) two RT-PCR protocols.
40X MultiScribe Reverse Transcriptase/RNase Inhibitor Mix (250 L) DESCRIPTION QUANTITY PART NO.
TaqMan One-Step RT-PCR 4310299 TaqMan Gold RT-PCR Kit 200 reactions N808-0232
Master Mix Reagents Protocol Contains
TaqMan PCR Core Reagents Kit (N808-0228)
10-pack, TaqMan One-Step 2000 Reactions 4313803
TaqMan Reverse Transcription Reagents (N808-0234)
RT-PCR Master Mix Reagents
TaqMan Gold RT-PCR Kit Protocol 402876
TaqMan EZ RT-PCR Core Reagents 10-pack, TaqMan Gold RT-PCR Kit 2000 reactions 4304133
The TaqMan EZ RT-PCR Core Reagents combine rTth DNA Polymerase
TaqMan Gold RT-PCR Kit With Controls 200 reactions N808-0233
with the fluorogenic 5' nuclease assay in a one-step RT-PCR format. This
Contains
format is ideally suited to high sample throughput and provides the addi- TaqMan PCR Core Reagents Kit (N808-0228)
tional benefit of high-temperature reverse transcription, which can prove TaqMan Reverse Transcription Reagents (N808-0234)
beneficial when amplifying targets in regions of RNA with high levels of TaqMan Human GAPDH Control Reagents (402869)
secondary structure. Protocol (402876)
DESCRIPTION QUANTITY PART NO. TaqMan Reverse Transcription Reagents 200 reactions N808-0234
TaqMan EZ RT-PCR Core Reagents 200 reactions N808-0236 Contains
Contains MultiScribe Reverse Transcriptase, 50 U/L, 100 L, 5000 units total
rTth DNA Polymerase, 2 tubes, each tube 2.5 U/L, 200 L, 500 units RNase Inhibitor, 2 tubes, each tube 20 U/L, 100 L, 2000 units
5X TaqMan EZ Buffer, 2 tubes, each tube 1.0 mL, 250 mM Bicine, 575 mM K(OAc)2, dNTP Mixture, 1 mL, 2.5 mM each dATP, dCTP, dGTP, and dTTP
0.05 mM EDTA, Passive Reference 1, 40% (w/v) glycerol, pH 8.2 Random Hexamers, 100 L, 50 M
AmpErase UNG, 1 U/L, 100 L,100 units total Oligo d(T)16, 100 L, 50 M
dATP, dCTP, and dGTP, 320 L,10 mM each 10X RT Buffer, 1.5 mL containing 500 mM KCl, 100 mM Tris-HCl, pH 8.3
dUTP, 320 L, 20 mM MgCl2 solution, 1.5 mL, 25 mM
Mn(OAc)2 solution, 2 tubes, 1 mL each, 25 mM 10-pack, TaqMan Reverse 2000 reactions 4304134
Transcription Reagents

Choices for TaqMan RT-PCR

TaqMan One-Step RT-PCR TaqMan Gold RT-PCR Kit TaqMan EZ RT-PCR
Master Mix Reagents Kit Core Reagents
RT Enzyme MultiScribe Reverse Transcriptase MultiScribe Reverse Transcriptase rTth DNA Polymerase
PCR Enzyme AmpliTaq Gold DNA Polymerase AmpliTaq Gold DNA Polymerase rTth DNA Polymerase
RT Temperature Low (48C) Low (48C) High (60C)
RT Primers Gene-specific primers only Gene-specific primers Gene-specific primers only
Random hexamers
Oligo dT
Number of steps One-step only One-step or two-step (flexible) One-step only

AmpErase UNG compatible No Yes, with two-step protocol only Yes
RT minus control Yes Yes Substitute MgCl2
Recommended for.... One-step RT-PCR assays not Evaluating between a one-step One-step RT-PCR assays that
requiring a high temperature RT and two-step RT-PCR protocol benefit from high-temperature
reverse transcription
SYBR Green kits
SYBR Green PCR Core Reagents
DESCRIPTION QUANTITY PART NO. The incorporation of SYBR Green 1 dye into a real-time PCR reaction
SYBR Green PCR Core Reagents 200 reactions 4304886 allows the detection of any double-stranded DNA generated during PCR.
Contains This provides great flexibility because no target specific probes are
AmpliTaq Gold DNA Polymerase. 5 U/L, 50 L, 250 units total
required, however both specific and non-specific products will generate
10X SYBR Green Buffer, 1.5 mL, contains SYBR Green 1 dye, Passive Reference 1
and optimized buffer components (proprietary formulation) signal. The use of the hot-start enzyme AmpliTaq Gold DNA Polymerase in
dNTP blend with dUTP. The single vial blend contains 1 mL of a 12.5 mM solution all SYBR Green 1 reagent kits allows the highest performance available
(2.5mM each of dATP, dCTP, dGTP and 5.0 mM of dUTP) by minimizing non-specific product formation. SYBR Green 1 assays are
MgCl2, 2 tubes, 1.5 mL each, 25 mM
ideal for use in target identification (screening assays), or when only a
SYBR Green PCR Core Reagents Protocol 4304965 small number of reactions are required for a given assay.
10-pack, SYBR Green PCR 2000 reactions 4306736
Core Reagents
SYBR Green PCR Master Mix
SYBR Green PCR Master Mix contains all the necessary components to
SYBR Green 1 assay
perform real-time PCR analysis in a convenient and easy-to-use premix.
Input sample should be DNA or cDNA. Recommended storage conditions
are 2-8C, so no thawing is required before use.
DESCRIPTION QUANTITY PART NO.
SYBR Green PCR Master Mix 200 reactions 4309155
1 bottle, 5 mL SYBR Green 1 dye fluoresces when bound to double-stranded DNA.
Contains
SYBR Green 1 dye, AmpliTaq Gold DNA Polymerase, dNTPs (with dUTP),
Passive Reference 1, and optimized buffer components (proprietary formulation)
SYBR Green PCR Master Mix Protocol 4310251
10-pack, SYBR Green PCR 2000 reactions 4312704
Master Mix
SYBR Green RT-PCR Kit
The SYBR Green RT-PCR Kit is a flexible kit enabling a one-step proto-
col for easy reaction set-up, or a two-step protocol for detection of multi- When DNA is denatured, the SYBR Green 1 dye is released and fluorescence is
ple targets from a single cDNA synthesis.
drastically reduced.
DESCRIPTION QUANTITY PART NO.
SYBR Green RT-PCR Reagents 200 reactions 4310179
Contains
SYBR Green PCR Master Mix (4309155)
TaqMan Reverse Transcription Reagents (N808-0234)

SYBR Green PCR Master Mix Protocol 4310251

During extension phase, primers anneal and PCR product is generated.

Polymerization is complete and SYBR Green 1 dye binds, resulting in a net increase
in fluorescence.

*Purchase of this product is accompanied by a limited license under U.S. Patent 5,035,996 and foreign equivalents to use for research.
 human
cytokine card
TaqMan Human Cytokine Card allows data to be quickly analyzed and incorporated into cytokine expres-
sion profile plots (Figure 3), which display cytokine expression levels of
unknown samples relative to a calibrator sample. All assays on the
TaqMan Human Card are RNA specific (cDNA must be loaded onto the
card) and are optimized for use with TaqMan Universal PCR Master Mix.
1E+04

1E+03

1E+02

Relative Expression
1E+01

1E+00

1E-01

1E-02

Fig. 1: The TaqMan Human Cytokine Card comes complete with an

IL-12p35
IL-12p40

GM-CSF
M-CSF
G-CSF
attached sample loading port.

IL-10

IL-13
IL-15
IL-17
IL-18

TNFb
IL-1b

TGFb
TNFa
IL-1a

IFNg
IL-2
IL-3

IL-8
IL-6
IL-7
IL-5
IL-4

LT-b
The TaqMan Human Cytokine Card represents the first product to use Legend: 0 hr 2 hr 6 hr 24 hr
the ABI PRISM Card, an exciting new consumable format allowing 1 L
reaction volumes, combined with incredible ease and accuracy of load- Fig. 3: Expression profile generated from aliquots taken from a single sample at 4
ing. The TaqMan Human Cytokine Card assays a single sample for 24 separate time points: before activation (calibrator), at 2 hours, 6 hours and 24
cytokine targets using 18S ribosomal RNA as an endogenous control. hours after activation.
Sample loading is performed in an ABI PRISM Card Filling Station via a
vacuum loading process and a sample loading consumable attached to The TaqMan Human Cytokine Card Upgrade Package enables:
the card (Figure 1). A series of connected channels (Figure 2) distributes Reduced reaction volumes resulting in reduced running costs
a sample-specific reaction mix to all 96 wells. The total volume required Simple and rapid sample loading
to load the TaqMan Human Cytokine Card is only 250 L, compared to Profiling of 24 human cytokines
the 5 mL required to load a traditional 96-well reaction plate (50 L
Rapid data analysis with the ABI PRISM Relative Quantification Software
reactions). The new ABI PRISM 7700 Relative Quantification Software
List of targets:
IL-1a IL-1 IL-2 IL-3 IL-4 IL-5
IL-6 IL-7 IL-8 IL-10 IL-12p35 IL-12p40
IL-13 IL-15 IL-17 IL-18 G-CSF GM-CSF
GM-CSF IFN-g LT- TGF- TNF-a TNF-

DESCRIPTION PART NO.

TaqMan Cytokine Card Upgrade Package 4311899
TaqMan Cytokine Gene Expression Cards (box of 10) 4310832

For Research Use Only. Not for use in diagnostic procedures.

Fig. 2: Interconnected channels allow rapid and even distribution of sample-

specific reaction mix to all 96 wells.

For the latest information on new ABI PRISM Card products covering new target categories, visit the
Sequence Detection Systems home page at: www.appliedbiosystems.com/sds


TaqMan Cytokine Gene

Expression Plate/PDARs
TaqMan Cytokine Gene Expression Plate TaqMan Pre-Developed Assay Reagents (PDARs) for
Gene Expression Quantification
The TaqMan Cytokine Gene Expression Plate offers a subset of the
assays available on the TaqMan Cytokine Gene Expression Card in the Researchers can now conduct experiments to determine the relative
conventional 96-well plate format. The plate contains primers and quantification of gene expression for a variety of gene sequences using
TaqMan probes for twelve human cytokine targets loaded and dried in TaqMan PDARs. TaqMan PDARs use the 5 nuclease assay to quantitate
an optical 96-well reaction plate (each individual cytokine assay is target and control sequences in cDNA samples, and are conveniently
loaded into 8 wells). In addition, every well on the plate contains the supplied as primer and TaqMan probes mixes optimized for use with
primers and TaqMan probe for the detection of 18S ribosomal RNA. TaqMan Universal PCR Master Mix. All TaqMan PDARs use identical
Relative expression profiles, in which expression levels are calculated thermal cycling parameters on the ABI PRISM 7700 and GeneAmp
relative to a calibrator sample, can easily be generated with the ABI 5700 Sequence Detection Systems, allowing multiple targets to be
PRISM 7700 Relative Quantification Software (Figure 4). All assays on assayed on the same plate. TaqMan PDAR endogenous control assays
the The TaqMan Cytokine Gene Expression Plate 1 (2 plates per kit) are enable relative gene expression studies to be performed. When perform-
RNA specific (cDNA must be loaded onto the plate), and are optimized ing such studies gene expression values are expressed relative to a
for use with TaqMan Universal PCR Master Mix. calibrator sample, eliminating the need to run standard curves, and
1E+05 resulting in an increase in throughput.
1E+04
TaqMan PDARs for Allelic Discrimination
Relative Expression

1E+03

1E+02 TaqMan PDARs for Allelic Discrimination (TaqMan PDARs for AD) use
the 5 nuclease assay to genotype purified DNA samples for specific
1E+01
polymorphisms and mutations. Most TaqMan PDARs for AD discriminate
1E+00 between two alleles differing by a single nucleotide polymorphism (SNP).
Each assay contains two different TaqMan probes (each labeled with a
1E-01
unique reporter dye) which bind preferentially to one of the alleles. The
IL-12p40
IL-12p35

TaqMan probes contained in the TaqMan PDARs for AD incorporate a

IL-10

IL-15
IL-1b

TNFa
IFNg
IL-2

IL-4

IL-8
IL-5
IL-a

minor groove binder (MGB) which enhances the melting temperature of

Legend: 0 hr 2 hr 6 hr 24 hr the probe, thus allowing shorter probes to be used. Using shorter probes
for allelic discrimination assays results in better discrimination between
Fig. 4: Expression profile generated from aliquots taken from a single sample at 4 matched and mismatched probes, producing a more robust assay.
separate time points: before activation (calibrator), and at 2 hours, 6 hours and 24
hours after activation.

List of targets:
IL-1a IL-1 IL-2 IL-4 IL-5 IL-8 For the latest information on TaqMan PDARs covering
IL-10 IL-12p35 IL-12p40 IL-15 IFN-g TNF-a gene expression quantification and allelic discrimination,
visit the TaqMan PDAR list on the Applied Biosystems website at:
DESCRIPTION PART NO.
TaqMan Cytokine Gene Expression Plate 1 4307266 www.appliedbiosystems.com/pdarlist
with TaqMan Universal PCR Master Mix,
Human Control RNA and Protocol
TaqMan Cytokine Gene Expression Plate 1 4307265
with TaqMan Universal PCR Master Mix
TaqMan Cytokine Gene Expression Plate 1 Protocol 4306744
 TaqMan Human Endogenous

TaqMan Human Endogenous Control Plate DESCRIPTION PART NO.

The amplification of an endogenous control can be performed to TaqMan Human Endogenous Control Plate with 4309920
normalize the amount of sample RNA or DNA added to a reaction. For TaqMan Universal PCR Master Mix,
the quantitation of gene expression, researchers commonly use -actin, Human Control RNA and Protocol
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal RNA TaqMan Human Endogenous Control Plate with 4309921
(rRNA). Deciding upon a specific control can be difficult however, even TaqMan Universal PCR Master Mix
when detailed information about the assay system is available. This can TaqMan Human Endogenous Control Plate Protocol 4308134
result in trial and error being required to identify an appropriate control,
leading to project delays and increased costs.
Targets
The TaqMan Human Endogenous Control Plate is a new tool designed
Targets included on the TaqMan Human Endogenous Control Plate are
to identify an endogenous control for gene expression studies. The
conveniently available as individual assays once an appropriate
Endogenous Control Plate offers the following:
endogenous control assay has been selected. These targets are available
Simultaneous evaluation of eleven candidate endogenous control assays
as either SDS Control Kits or as TaqMan Pre-Developed Assay Reagents
Cost effective endogenous control identification
(PDARs).
Easy plate set-up with pre-loaded TaqMan probe and primer sets

Internal Positive Control (IPC) assay to identify PCR inhibition Note: The same 18S rRNA TaqMan probe and primer set is available as
both an SDS Control Kit and a PDAR, with the products differing only in
The TaqMan Human Endogenous Control Plate consists of an Optical configuration.
96-Well Reaction Plate (2 per kit) loaded with TaqMan probes and
primers for eleven candidate control assays and an IPC assay (arranged SDS Control Kits
into twelve columns of eight wells). TaqMan Universal PCR Master Mix DESCRIPTION QUANTITY PART NO.
and MicroAmp Optical Caps are included. TaqMan Ribosomal RNA 1000 reactions 4308329
IE+01
Control Reagents
Contains
rRNA TaqMan Probe [VIC], 250 L, 40 M
IE+00 rRNA Forward Primer, 250 L, 10 M
Relative Expression

rRNA Reverse Primer, 250 L, 10 M

Total RNA Control, 100 L, 50 ng/mL
IE-01
TaqMan Ribosomal RNA 4308310
Control Reagents Protocol
IE-02 TaqMan Exogenous Internal 200 reactions 4308323
Positive Control Reagents
Contains
huHPRT
huB2m

huGUS
huPGK

huTBP
huCYC

huTfR
huGap
huPO

hubA

10X Exogenous IPC Mix, 1.0 mL

18S
IPC

10X Exogenous IPC Block, 120 L

Legend: 1 2 3 4 5 6 7 8
50X Exogenous IPC DNA, 200 L
Fig. 1: Results from the TaqMan Human Endogenous Control Plate shown as a gene TaqMan Exogenous Internal 4308335
expression profile. Results are for eight samples in total with sample 1 designated Positive Control Reagents Protocol
as the calibrator. 5-pack, TaqMan Exogenous Internal 1000 reactions 4308321
Positive Control Reagents
Data generated using the TaqMan Human Endogenous Control Plate can TaqMan Exogenous Internal 200 reactions 4308320
be viewed as relative expression profiles (Figure 1). Expression profiles Positive Control Reagents with
for each sample are expressed relative to the calibrator sample for all TaqMan Universal PCR Master Mix
twelve assays (the expression level of the calibrator is designated as Contains
zero). In this example the expression of 18S rRNA is stable for all eight TaqMan Exogenous Internal Positive Control Reagents (4308323)
samples (showing minimal deviation from the calibrator), making this TaqMan Universal PCR Master Mix (4304437)
assay a suitable choice for an endogenous control.
Control Plate
PDARs* Components
The following targets are available as TaqMan PDARs. They are supplied DESCRIPTION QUANTITY PART NO.
as a 20X primer and probe mix (sufficient for 1,000 reactions of 50 L
Sequence Detection Systems 4305822
each) and are optimized for use with TaqMan Universal PCR Master Mix
Spectral Calibration Kit
in the second step of a two-step RT-PCR reaction.
Contains:
DESCRIPTION PART NO. 7700 dye calibration standards for FAM, TET, VIC, JOE,
18S 18S Ribosomal RNA 4310893E SYBR Green 1, TAMRA and ROX

huPO Acidic ribosomal protein (human) 4310879E MultiScribe Reverse 100 L, 50 U/L 4311235
Transcriptase
huA Beta-actin (human) 4310881E**
RNase Inhibitor 100 L, 20 U/L N808-0119
huCYC Cyclophilin (human) 4310883E
AmpErase UNG
100 L, 1 U/L N808-0096
huGAPDH Glyceraldehyde-3-phosphate 4310884E**
dehydrogenase (human) Random Hexamers 100 L, 50 M N808-0127

huPGK Phosphoglycerokinase (human) 4310885E Oligo d(T)16 100 L, 50 M N808-0128

hu2m 2-Microglobulin (human) 4310886E

GeneAmp dNTP Blend 1 mL, 10 mM N808-0260
(2.5 mM each dNTP)
huGUS -Glucronidase (human) 4310888E
GeneAmp dNTP Blend 1 mL, 12.5 mM N808-0270
huHPRT Hypoxanthine ribosyl transferase (human) 4310890E (with dUTP) (2.5 mM each of
dATP, dCTP, dGTP,
huTBP Transcription factor IID, TATA binding 4310891E
and 5.0 mM dUTP)
protein (human)
Control Total RNA (Human) 100 L at 50 ng/L 4307281
huTfR Transferrin receptor (human) 4310892E
Control DNA (Human) 2 tubes, each tube 4312660
**These assays do not amplify the same amplicon as the TaqMan -actin Detection Reagents (P/N 100 L at 50 ng/L
401846) and the TaqMan Human GAPDH Control Reagents (P/N 402869). They have been
redesigned and improved for the TaqMan Human Endogenous Control Plate. 20% Glycerol Solution, 100 mL 402929
Molecular Biology Grade

*Purchase of this product is accompanied by a limited license under U.S. Patent 5,035,996 and foreign equivalents to use for research.
 optical
consumables
Designed for Fluorescent Applications MicroAmp Optical Caps
MicroAmp Optical Caps have a thinner dome than traditional MicroAmp
MicroAmp and ABI PRISM Optical consumables are the only plastic Caps to allow more efficient light transmission. MicroAmp Optical Caps
consumables designed and optimized for Sequence Detection Systems can be used with Optical Tubes and Optical 96-Well Reaction Plates.
instrumentation. All consumable plastics are quality controlled to DESCRIPTION QUANTITY PART NO.
minimize fluorescent background. Optical tubes and plates are frosted MicroAmp Optical Caps, 300 strips N801-0935
to minimize contaminating fluorescence from wells of the thermal 8 caps/strip 2400 caps
cycling block.
MicroAmp Optical Tubes
ABI PRISM Optical Adhesive Covers MicroAmp Optical Tubes can be used used with the ABI PRISM 7700 and
ABI PRISM Optical Adhesive Covers have been designed as an easy-to-use GeneAmp 5700 Sequence Detection Systems and must be used with a
alternative to MicroAmp Optical Caps when using the ABI PRISM 7700 MicroAmp Tray/Retainer Set.
and GeneAmp 5700 Sequence Detection Systems. Excellent optical DESCRIPTION QUANTITY PART NO.
clarity combined with low fluorescent background make Optical Adhesive MicroAmp Optical Tubes 2000 N801-0933
Covers ideal for both real-time and end-point applications. Optical
Adhesive Covers require the use of an ABI PRISM Optical Cover MicroAmp Tray/Retainer Set
Compression Pad and are designed for use with ABI PRISM 96-Well To be used with MicroAmp Optical Tubes.
Optical Reaction Plates.
DESCRIPTION QUANTITY PART NO.
DESCRIPTION QUANTITY PART NO. MicroAmp Tray/Retainer Set 10 sets 403081
ABI PRISM Optical Adhesive Each 4313663
Cover Starter Kit
Contains
ABI PRISM Optical Adhesive Covers (quantity 20)
Applicator (quantity 1)
ABI PRISM Optical Cover Compression Pad (quantity 1)
L IGHT B EAM
ABI PRISM Optical Adhesive Covers 100 4311971
ABI PRISM Optical Cover 5 4312639 Optical Tubes and Optical 96-well Reaction
Compression Pads Plates remain closed throughout the PCR
ABI PRISM Optical Adhesive Covers 100 4314320 amplification and detection process,
and 96-Well Optical Reaction Plates thereby reducing the chance
Contains
ABI PRISM Optical Adhesive Covers (4311971)
of contamination.
5X ABI PRISM Optical 96-Well Reaction Plates (4306737)

ABI PRISM Optical 96-Well Reaction Plate with Barcode

The ABI PRISM Optical 96-Well Reaction Plate is constructed from a
single rigid piece of polypropylene in a convenient 96-well microplate
format, and is supplied complete with a barcode (code 128) for easy
sample tracking. L ENS
DESCRIPTION QUANTITY PART NO.
ABI PRISM Optical 96-Well 20 plates 4306737
Reaction Plates with Barcodes C AP
ABI PRISM Optical 96-Well Reaction 20 plates 403012
Plates and MicroAmp Optical Caps 2400 caps T UBE
Contains
N801-0935 and 4306737

T HERMAL C YCLER B ASE

Custom Synthesis
Services
Rapid Assay Development Guidelines
A key component of the Rapid Assay Development Guidelines (see
Introduction page) is the design of primers and probes. The following
primer and probe design recommendations will ensure success when
used within the Rapid Assay Development Guidelines.
Synthesis Services
Custom ABI PRISM TaqMan Probes can be synthesized by the Applied
Biosystems Custom Oligonucleotide Synthesis Service. TaqMan probes
are available in three different sizes, with a variety of dyes, and are HPLC
purified.
CUSTOM SYNTHESIZED TAQMAN PROBES

When designing TaqMan probes: DESCRIPTION NO. REACTIONS* DYES AVAILABLE PART NO.
Keep the G/C content in the range of 20% to 80%. 50,000 ~10,000 5 Fluorescent label: 450003
Avoid runs of identical nucleotides (especially guanine, where runs of 100,000 pmol 6-FAM, VIC or TET.
four or more Gs should be avoided). 3 label: TAMRA
No G on the 5 end. 15,000 ~2,500 5 Fluorescent label: 450024
Keep the melting temperature (Tm) in the range of 6870C for 25,000 pmol 6-FAM, VIC or TET.
quantitation, 6567C for allelic discrimination when using Primer 3 label: TAMRA
Express software. 5,000 ~500 5 Fluorescent label: 450025
Select the strand that gives the probe more Cs than Gs.
6,000 pmol 6-FAM, VIC or TET.
3 label: TAMRA
For allelic discrimination:
* Based on final reaction volume of 50 L, 200 nM probe concentration.
Position the polymorphism site approximately in the center of
the probe.
SEQUENCE DETECTION PRIMERS (UNLABELED)
Adjust the probe length so that both probes have the same Tm.
QUANTITY PART NO.
When designing sequence detection primers: Minimum 130,000 pmols 4304972
Short amplicons work best. Try to keep the amplicon size in the range of Minimum 40,000 pmols 4304971
50150 bps. Minimum 4,000 pmols 4304970
Keep the G-C content in the range of 20% to 80%.
Avoid runs of identical nucleotides (especially guanine, where runs of
four or more Gs should be avoided).
Keep the melting temperature (Tm) in the range 5860C when using
Primer Express software.
The five nucleotides at the 3 end should include no more than two
G and/or C bases.
Place the forward and reverse primers as close as possible to the probe
without overlapping the probe.
Use an annealing/extension temperature of 60C for quantitation, 62C
for allelic discrimination (except TaqMan PDARs for Allelic
Discrimination).

Dye Combination Guide

ABI PRISM TaqMan Probes can be synthesized with different reporter
dyes and combined in a single reaction on the ABI PRISM 7700
Sequence Detection System. The dye combinations listed below are
recommended for two-probe applications such as relative quantitation,
allele detection, and plus/minus detection.
TAQMAN PROBE DYE RECOMMENDATIONS BY APPLICATION
APPLICATION REPORTER DYE
First Probe Second Probe
Relative Quantitation [FAM] [VIC]
Allele Detection [FAM] [VIC]
Plus/Minus [FAM] [VIC]
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on six continents. For international
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headquarters or refer to our web site
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information for life scientists. PE
Corporation consists of the Applied
Biosystems and Celera Genomics
businesses.

Headquarters
850 Lincoln Centre Drive
Foster City, CA 94404 USA
Phone: 650.638.5800
Toll Free: 800.345.5224
Fax: 650.638.5884

For Research Use Only.

Not for use in diagnostic procedures.

Certain Applied Biosystems PCR reagents

are developed and manufactured by
Roche Molecular Systems, Inc.,
Branchburg, New Jersey, U.S.A.

The PCR and 5' nuclease process are covered by patents

owned by Roche Molecular Systems, Inc. and F. Hoffmann-La
Roche Ltd. AmpErase, AmpliTaq Gold, Gene Amp, and
TaqMan are registered trademarks of Roche Molecular
Systems, Inc. The SYBR Green dye is sold pursuant to a lim-
ited license from Molecular Probes Inc. SYBR is a registered
trademark of Molecular Probes Inc. ABI PRISM and its design,
Applied Biosystems, MicroAmp and Primer Express are regis-
tered trademarks and FAM, MultiScribe, TAMRA, and VIC are
trademarks of PE Corporation or its subsidiaries in the U.S.
and certain other countries.

Applied Biosystems develops and manufactures

its products in accordance with ISO 9000 quality
systems requirements.

*Purchase of this product is accompanied by a limited

license under U.S. Patent 5,035,996 and foreign equivalents
to use for research.

Information subject to change without notice.

2000 Applied Biosystems. All rights reserved.

Specifications subject to change without notice
Printed in the USA. Order No. 790102-003

www.appliedbiosystems.com