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A4251K

Instruction manual THUNDERBIRD SYBR qPCR Mix 2004

THUNDERBIRDTM SYBRTM qPCR Mix

QPS-201T
QPS-201

1 mL x 1
1.67 mL x 3

Store at -20°C, protected from lightContents[1]

Introduction[2]

Components[3]

Primer design[4]Template DNA[5]

Protocol
1. Standard reaction set up
2. Cycling condition
2-1. Real-time PCR conditions using Applied Biosystems 7900HT
2-2. Real-time PCR conditions using Roche LightCycler 1.1
2-3. 3-step cycle[6]

Related Protocol
cDNA synthesis[7]

Troubleshooting[8]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe generallaboratory precaution and utilize safety while using this kit.
This product is sold in U.S. under the license of US patent 7772383 from Chakrabarti Advanced Technology NewCo LLC.
-LightCycler™ is a trademark of Idaho Technology, Inc. and Roche Molecular Systems, Inc.
-SYBR® is a registered trademark of Molecular Probes, Inc.

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www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
THUNDERBIRDTM SYBR® qPCR Mix is a Taq DNA polymerase-based highly efficient
2x Master Mix for real-time PCR using SYBR® Green I. The master mix contains all
required components, except ROX reference dye and primers (50x ROX reference dye is
individually supplied with this kit). The master mix facilitates reaction setup, and improves
the reproducibility of experiments.
This product is an improved version of Realtime PCR Master Mix (Code No. QPK-201)
and Realtime PCR Master Mix -Plus- (Code No. QPK-212). In particular, the reaction
specificity and PCR efficiency is enhanced.

Features-High specificity
The specificity for the detection of low-copy targets is improved.
-Homogeneous amplification
The dispersion of PCR efficiency between targets is reduced by a new PCR enhancer*.
(*Patent pending)-Broad dynamic range
High specificity and effective amplification enable the detection of a broad dynamic range.
-Compatibility for various real-time cyclers.
The reagent is applicable to most real-time cyclers (i.e. Block type and glass capillarytype). Because the 50x ROX reference dye is individually supplied with this kit, the kit
can be applied to real-time cyclers that require a passive reference dye.
-Hot start PCR
The master mix contains anti-Taq DNA polymerase antibodies for hot start technology.
The antibodies are easily inactivated in the first denaturation step, thereby activating the
DNA polymerase.

About the SYBR® Green I detection system
The SYBR® Green I assay system utilizes fluorescent emission when SYBR® Green isintercalated into double-stranded DNA. The signal depends on the amount of amplified
DNA. However, this system cannot distinguish between target and non-specific amplicons.
Therefore, melting curve analysis is necessary after amplification.

[ 2 ] Components

This kit includes the following components for 40 reactions (QPS-201T) and 200 reactions(QPS-201), with a total of 50 μL per reaction. All reagents should be stored at -20 °C.
<QPS-201T>
THUNDERBIRDTM SYBR® qPCR Mix
50xROX reference dye

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1 mL x 1
50 μL x 1

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qPCR Mix
1.67 mL x 3
50xROX reference dye

250 μL x 1

Notes:-THUNDERBIRDTM SYBR® qPCR Mix can be stored, protected from light, at 2-8°C for
up to 3 months. For longer storage, this reagent should be kept at -20°C and protectedfrom light. No negative effect was detected by 10 freeze-thaw cycles of
THUNDERBRID™ SYBR® qPCR Mix. This reagent does not contain the ROX referencedye.
-50x ROX reference dye can be stored, protected from light, at 2-8°C or -20°C. For realtime cyclers that require a passive reference dye, this reagent must be added to the reaction
mixture at a concentration of 1x or 0.1x. The master mix solution with the ROX referencedye can be stored, protected from light, at 2-8°C for up to 3 months. For longer storage,
this reagent should be kept at -20°C and protected from light. The pre-mixed reagents canbe prepared according to the following ratios. [5] Table 1 shows the optimal concentrationof the ROX dye.
1x Solution
THUNDERBIRDTM SYBR® qPCR Mix : 50xROX reference dye = 1.67 mL : 66.8 μL
THUNDERBIRDTM SYBR® qPCR Mix : 50xROX reference dye = 1 mL : 40 μL
0.1x Solution
THUNDERBIRDTM SYBR® qPCR Mix : 50xROX reference dye = 1.67 mL : 6.7 μL
THUNDERBIRDTM SYBR® qPCR Mix : 50xROX reference dye = 1 mL: 4 μL
For real-time cyclers that do not require a passive reference dye, THUNDERBIRDTM
SYBR® qPCR Mix without the ROX reference dye can be used.

[ 3 ] Primer design
1. Primer conditions
Highly sensitive and quantitative data depend on primer design. The primer should bedesigned according to the following suggestions;-Primer length: 20-30 mer-GC content of primer: 40-60%-Target length: ≤ 200 bp (optimally, 80-150 bp)-Melting temperature (Tm) of primers: 60-65°C-Purification grade of primers: Cartridge (OPC) grade or HPLC grade
Notes:-Longer targets (>200 bp) reduce efficiency and specificity of amplification.
-Tm of the primers can be flexible, because the Tm value depends on the calculation
formula.
<QPS-201>
THUNDERBIRDTM SYBR®
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www.toyobo.co.jp/e/bio[ 4 ] Template DNA

The following DNA samples can be used as templates.

1. cDNA
Non-purified cDNA, generated by reverse transcription reactions, can be used directly forreal-time PCR using THUNDERBIRDTM SYBR® qPCR Mix. Up to 10% of the volume of
a cDNA solution can be used for a real-time PCR reaction. However, excess volume of thecDNA may inhibit the PCR. Up to 20% (v/v) of the cDNA solution from ReverTra AceTM
qPCR RT Kit (Code No. FSQ-101) can be used for real-time PCR.

2. Genomic DNA, Viral DNA
Genomic DNA and viral DNA can be used at up to 200 ng in 50 μL reactions.

3. Plasmid DNA
Although super-coiled plasmids can be used, linearized plasmid DNA produces moreaccurate assays. The copy number of the plasmid DNA can be calculated by the following
formula.
Copy number of 1µg of plasmid DNA = 9.1 x 1011 / Size of plasmid DNA (kb)

[ 5 ] Protocol

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1. Reaction mixture setup

Reaction volume

Final

Reagent
DW

50μL
X μL

20μL
X μL

Concentration

THUNDERBIRDTM SYBR® qPCR Mix

25 μL

10 μL

1x

Forward Primer
Reverse Primer

15 pmol
15 pmol

6 pmol6 pmol

0.3 μM*1
0.3 μM*1

50X ROX reference dye

1μL / 0.1μL 0.4μL / 0.04μL 1x / 0.1x*2

DNA solution
Total

Y μL
50 μL

Y μL
20 μL

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Notes:*1 Higher primer concentration tends to improve the amplification efficiency, and lower
primer concentration tends to reduce the non-specific amplification. The primer
concentration should be set between 0.2-0.6 µM.
*2 50xROX reference dye must be added when using real-time cyclers that require a
passive reference dye, according to Table 1. Table 1 shows the optimum concentrationof the ROX reference dye. This dye is not necessary for real-time cyclers that do not
require a passive reference dye.
Table 1 Recommended ROX dye concentration
Real-time cycler
Applied Biosystems 7000, 7300, 7700, 7900HT
StepOne™, StepOnePlus™ etc.
Applied Biosystems 7500, 7500Fast,
Stratagene cyclers (Option) etc.
Roche cyclers, Bio-Rad cyclers, BioFlux cyclers etc.

Optimal dye concentration(dilution ratio)
1x (50:1)
0.1x (500:1)
Not required

Notes:The ROX dye in SYBR® Green Realtime PCR Master Mix (Code No. QPK-201) andSYBR® Green Realtime PCR Master Mix -Plus- (Code No. QPK-212) correspond to 1x
concentration.

2. PCR cycling conditions
The following table shows the recommended thermal conditions using primers designed
according to the recommended primer described in [3]. Almost all targets can also be
amplified using the ongoing conditions with other real-time PCR reagents.
When satisfactory results are not obtained or the Tm value of the primers is lower than60°C, a 3-step cycle should be used. The detailed conditions are described in [5] 2-3.

<2-step cycle>

Temperature

Time

Ramp

Pre-denaturation

95°C

20-60 sec.*1.

Maximum

95°C

1-15 sec.*2.

Maximum

Denaturation:

60°C*330-60 sec.*4.
Maximum(data collection should be set at the extension step)
Melting / Dissociation Curve Analysis*5
Extension:

*1

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40 cycles

Due to the anti-Taq antibody hot start PCR system, the pre-denaturation can becompleted within 60 sec. The pre-denaturation time should be determined according to
the recommendations of each real-time cycler. If the optimal pre-denaturation timecannot be determined, the time should be set at 60 sec.

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Table 2 The recommended pre-denaturation time on each real-time cyclers
Real-time cycler
Pre-denaturation timeHigh speed cycler (e.g. Applied Biosystems 7500Fast)
20 secCapillary cycler (e.g. Roche LightCycler™ 1.x, 2.0)
30 secGeneral real-time cyclers (e.g. Applied Biosystems 7700,
60 sec7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers)*2 The following table shows the optimal denaturation times for each real-time cycler. If

the optimal denaturation time cannot be determined, the time should be set at 15 sec.
Table 3 The recommended denaturation time on each real-time cyclers
Real-time cycler
Pre-denaturation timeHigh speed cycler (e.g. Applied Biosystems 7500Fast)
3 sec
Capillary cycler (e.g. Roche LightCycler™ 1.x, 2.0)
5 sec
General real-time cyclers (e.g. Applied Biosystems 7700,
15 sec7500, 7900HT [normal block], StepOne™, StepOnePlus™
Agilent cyclers, BioFlux cyclers)*3 Insufficient amplification may be improved by decreasing the extension temperature,and non-specific amplification (e.g. abnormal shapes of the amplification curve at low
template concentrations) may be reduced by increasing the extension temperature. The
extension temperature should be set at 56-64°C.
*4 If the target size is smaller than 300 bp, the extension time can be set at 30 sec. on almost

all real-time cyclers. Instability of the amplification curve or variation of data from eachwell may be improved by setting the extension time at 45-60 sec. Some real-time cyclers
or software need over 30 sec. for the extension step. In these cases, the time should be set
according to each instruction manual (e.g. Applied Biosystems 7000/73000: ≥ 31 sec;
Applied Biosystems 7500: ≥ 35 sec.).
*5 The melting curve analysis should be performed according to the recommendations ofeach real-time cycler.

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2-1. Real-time PCR conditions using Applied Biosystems 7900HT(Normal block type, software version 2.2.2)
The following is an example of a SYBR® Green I assay using Applied Biosystems 7900HT.
(1) The cycling parameters should be set according to the following “Thermal Cycler
Protocol” window under the “Instrument” tab. When adding the melting curve
analysis, click the “Add Dissociation Stage” button.

Notes:- Appropriate sample volumes should be set.
- ≥ 45 sec. is necessary for the extension step.
(2) Click the “Data collection” tab.

(3)

Insert the PCR plate(4) Start the program

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conditions using Roche LightCycler 1.1(Software version 3.5)
The following is an example of a SYBR® Green I assay using Roche LightCycler 1.1.

2-2.

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The cycling parameters should be set according to the following window. Analysis
and Acquisition mode of the initial denaturation step must be set at “None”.

(2)

The cycling parameters should be set according to the following window. Analysis
mode of the PCR step must be set at “Quantification”. And Acquisition modes of
95°C and 60°C must be set at “None” and “Single”, respectively.

PCR
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tech_osaka@toyobo.jp(3)[Melting curve analysis] The cycling parameters should be set according to thefollowing window. The analysis mode must be set at “Melting curves”. Acquisition
modes of 95°C (first) and 65°C must be set at “Non”. Acquisition mode of thesecond 95°C must be set at “CONT”.

(4)

The cycling parameters should be set according to the following window. Analysis
and Acquisition mode of the cooling step must be set at “Non”.

(5)

Insert the capillaries in the carousel, and start the cycling program.

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2-3. 3-step cycle
In the event of the 2-step cycle failing, the following 3-step cycle may improve results. In
the following cases, the 3-step cycle conditions may improve the result efficiently.
-The Tm of the primer is lower than 60°C.
-The target is longer than 300 bp.
-PCR efficiency is low.

<3-step cycle>

TemperatureTime

Pre-denaturation

95°C

20-60 sec.*1.

Maximum

Denaturation:

95°C

1-15 sec.*1.

Maximum

Annealing

55-65°C*2

5-30 sec.*3

Maximum

Extension:

Ramp

40 cycles

30-60 sec.*4.

72°C(data collection should be set at the extension step)
Melting / Dissociation Curve Analysis*5*1

The denaturation step should be determined according to [5] 2.

*2

The annealing temperature should be set at primer’s Tm-5°C. A higher annealing
temperature may improve non-specific amplification.

*3 The annealing time should be set at 5 sec. (Fast cycler), 15 sec. (Normal cycler). Shorter

annealing times may reduce non-specific amplification. Longer annealing times (up to
30 sec.) may increase the PCR efficiency when the efficiency is low.

≤300 bp) require shorter extension times (≤30 sec.). However, certain
cyclers require >30 sec. detection time at the extension step. An unstable signal may be
improved by prolonging the extension time up to 40-60 sec. Also note that some cyclers
cannot have an extension time of 30 sec. (Applied Biosystems 7000, 7300: ≥ 31 sec,
7500: ≥35 sec.)*4 Shorter targets (

*5

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The melting curve analysis should be performed according to each cycler’s
recommendations.

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www.toyobo.co.jp/e/bio[ 6 ] Related Protocol

1. cDNA synthesis
cDNA synthesized by various cDNA synthesis reagents can be used with
THUNDERBIRDTM SYBR® qPCR Mix. However, cDNA synthesized by a reagent
specialized for real-time PCR can increase sensitivity.
ReverTra AceTM qPCR RT Kit (Code No. FSQ-101) is a cDNA synthesis kit suitable forreal-time PCR. Here, the protocol with ReverTra AceTM qPCR RT Kit is described.
However, for the detailed protocol, please refer to the instruction manual of the kit.
(1) Denaturation of RNA
Incubate the RNA solution at 65°C for 5 min and then chill on ice.
Notes:-This step can be omitted. But this step may increase the efficiency of the reverse
transcription of RNA, which forms secondary structures.
-Do not add 5x RT Buffer and/or enzyme solution at this step.
(2) Preparation of the reaction solution

Reagent
Nuclease-free Water
5x RT Buffer
Primer Mix
Enzyme Mix
RNA solution
Total

Volume (amount)
X μL
2 μL
0.5 μL0.5 μL0.5 pg-1 µg
10 μL(3) Reverse transcription reaction-Incubate at 37°C for 15 min. <Reverse transcription>-Heat at 98°C for 2 min. <Inactivation of the reverse transcriptase>-Store at 4°C or -20°C.**This solution can be used in the real-time PCR reaction directly or after dilution.
Notes:
The above temperature conditions are optimized for ReverTra AceTM qPCR RT Kit.

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www.toyobo.co.jp/e/bio[ 7 ] Troubleshooting
Symptom
Loss of linearity in thehigh cDNA/DNA
concentration region.

Cause

SolutionIntercalation of SYBR® Green I into
the template DNA.

Because SYBR® Green I is also intercalated into
the template DNA, the base line tends to behigher when high concentration DNA samples areused. Diluted template should be used to obtain acorrect Ct value.
When the DNA/cDNA copy number is lower than
10 copies per reaction, the linearity of the reaction
tends to be lost. The template concentration
should be increased.
The diluted DNA templates tend to be absorbed
onto the tube wall. Dilution should be performed
just prior to experiments.
Dimer formation may reduce the amplification
efficiency of the target, especially for reactions at
low template concentration. The reaction
condition should be optimized or the primer
sequences should be changed.
Non-specific amplification may reduce theamplification efficiency of the target. The reaction
conditions should be optimized or the primer
sequences should be changed.
Optimize the cycling conditions according to [5].
Fresh primer solution should be prepared.
The Ct value on the linear region should be used
to calculate PCR efficiency.
The Ct value on the linear region should be used
to calculate PCR efficiency.
Low-purity DNA may contain PCR inhibitors.
Re-purify the DNA samples.
Diluted DNA templates tend to be absorbed onto
the tube wall. Dilution of the template
DNA/cDNA should be performed just prior toexperiments.
In general, plasmid DNA or PCR product is used
at low concentration. Diluted DNA templates tend
to be absorbed onto the tube wall. Dilution of thetemplate DNA/cDNA should be performed justprior to experiments. Dilution with a carrier
nucleic acid solution (Yeast RNA) is also
effective in improving linearity.
Optimize the thermal conditions according to [5].
Different lots of primers may show different
results. When the lot is changed, prior testing of
the primer should be performed.

The template DNA is insufficient.

Lost of linearity or
lower signal in the low
DNA/cDNA
concentration region.

Adsorption of the DNA to the tube
wall.

Loss of linearity of theamplification carves.

Competition with non-specificamplification.

The PCR efficiency islower than 90% (slope:<-3.6)

Inappropriate cycling conditions.
Degradation of the primers.
The calculation of the PCR
efficiency is inappropriate.
The calculation of the PCR
efficiency is inappropriate.
Poor purification of the template
DNA
Absorption of the template DNA to
the tube wall.

The PCR efficiency ishigher than 110%.
Reproducibility is not
good.

Competition with primer dimer
formation.

Plasmid DNA or PCR product isused as a template.

Inappropriate thermal conditions.
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Symptom
Amplification from thenon-template control(NTC).

Cause
Formation of primer dimer.

Contamination or carry over of the
PCR products.

Low amplification
curve signal /Unstable amplification
curve signal.

Excessive amount of ROX referencedye.

Detection of multiple
peaks on the melting
curve analysis

Non-specific amplification.

Inappropriate settings offluorescence measurement
Insufficient reaction volume.

Formation of primer dimer.

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SolutionOn the melting curve analysis, a peak at atemperature lower than that of the target peaksuggests a primer dimer. The PCR cycle should be
optimized according to [4] (2). If the result is not
improved, the following should be
performed:change the primer sequence and/orchange the purification grade of the primer(HPLC grade)
When the no-template control generates a peak at
the same melting temperature as the target on themelting curve analysis, the amplification is caused
by a carry-over or contamination. Use fresh
reagents.
Excessive amount of ROX reference dye may
cause low signal. 50x ROX reference dye should
be used according to [5] Table 1.
Settings should be confirmed according to the
instruction manual of each detector.
Low reaction volume may cause an unstablesignal. Increase the reaction volume.
Optimize the reaction conditions. If the result is
not improved, the primer sequence should bechanged.
On the melting curve analysis, a peak at atemperature lower than that of the target peaksuggests a primer dimer. The PCR cycle should be
optimized according to [4] (2). If the result is not
improved, the following action should be
performed:change the primer sequence and/orchange the purification grade of the primer(HPLC grade)

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www.toyobo.co.jp/e/bio[ 8 ] Related products
Product name

Package
1mLx1
1.67mLx3
200 rxns

Code No.
QPS-101T
QPS-101
FSQ-101

High efficient cDNA synthesis master mix for real-time PCR
ReverTra AceTM qPCR RT Master Mix

200 rxns

FSQ-201

High efficient cDNA synthesis master mix for real-time PCR with genomic DNA remover
ReverTra AceTM qPCR RT Master Mix

200 rxns

FSQ-301

0.5 mL x 5

QRT-101

0.5 mL x 5

QRT-201

High efficient real-time PCR master mix
THUNDERBIRDTM Probe qPCR Mix
High efficient cDNA synthesis kit for real-time PCR
ReverTra AceTM qPCR RT Kit

with gDNA remover
One-step Real-time PCR master mix for probe assay

RNA-direct™ Realtime PCR Master Mix
One-step Real-time PCR master mix for SYBR® Green assay
RNA-direct™ SYBR® Realtime PCR Master Mix

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