www.toyobo.co.jp/e/bio

F1813K

Instruction manual THUNDERBIRD Next SYBR qPCR Mix 2103

THUNDERBIRDTM Next SYBR® qPCR Mix
QPX-201T
QPX-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® StepOnePlus™
2-2. Real-time PCR conditions using Roche LightCycler® 96
2-2. Real-time PCR conditions using Bio-Rad CFX96 Touch™ Deep Well
2-4. 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
general laboratory precaution and utilize safety while using this kit.
-LightCycler® is a registered trademark of Idaho Technology, Inc. and Roche Diagnostic Systems, Inc.
-SYBR® is a registered trademark of Thermo Fisher Scientific, Inc.
.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

1

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

Description
THUNDERBIRDTM Next SYBR® qPCR Mix is a highly efficient 2× Master Mix forreal-time PCR using SYBR® Green I. The master mix contains all required components,
except primers. The master mix facilitates reaction setup, and improves the
reproducibility of experiments.
This product is an improved version of THUNDERBIRDTM SYBR® qPCR Mix (CodeNo. QPS-201). 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.
-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 passive reference dye is included with this kit, the kit can be applied
to real-time cyclers that require a passive reference dye.
-Fast PCR
This reagent enables amplification using fast cycle condition.
-Hot start PCR
The master mix contains anti- DNA polymerase antibodies for hot start technology.
The antibodies are easily inactivated in the first denaturation step, thereby activating
the DNA polymerase.
-Utilization of dUTP
This master mix contains dUTP instead of dTTP. Therefore, the rate of false-positive
detection can be reduced by adding uracil-N-glycosylase (UNG). *UNG is not
supplied with this kit. Uracil-DNA Glycosylase (UNG), Heat-labile (Code No.
UNG-101) can be used.
-Utilization of a visible tracking dye
This master mix contains visible tracking dye. This dye helps to eliminate pipetting
errors, and does not spectrally overlap with fluorescent dyes used for qPCR and will
not interfere with real-time detection.

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-specificamplicons. Therefore, melting curve analysis is necessary after amplification.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

2

www.toyobo.co.jp/e/bio[ 2 ] Components

This kit includes the following components for 100 reactions (QPX-201T) and 500
reactions (QPX-201), with a total of 20 µL per reaction. All reagents should be stored at-20 °C.
<QPX-201T>
THUNDERBIRDTM Next SYBR® qPCR Mix

1 mL<QPX-201>
THUNDERBIRDTM Next SYBR® qPCR Mix

1.67 mL x 3

Notes:-THUNDERBIRDTM Next 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 andprotected from light. No negative effect was detected by 10 freeze-thaw cycles of
THUNDERBRIDTM Next SYBR® qPCR Mix.

[ 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: ≤ 500 bp (optimally, 80-200 bp)-Melting temperature (Tm) of primers: 60-65°C-Purification grade of primers: Cartridge (OPC) grade or HPLC grade
Notes:-Longer targets (>500 bp) reduce efficiency and specificity of amplification.
-Tm of the primers can be flexible, because the Tm value depends on the calculation
formula.

[ 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 Next SYBR® qPCR Mix. Up to 10% of thevolume of a cDNA solution can be used for a real-time PCR reaction. However, excess
volume of the cDNA may inhibit the PCR. Up to 20% (v/v) of the cDNA solution fromthe ReverTra AceTM qPCR RT Kit (Code No. FSQ-101), the ReverTra AceTM qPCR RT
Master Mix (Code No. FSQ-201) and the ReverTra AceTM qPCR RT Master Mix with
gDNA remover (Code No. FSQ-301) can be used for real-time PCR.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

3

www.toyobo.co.jp/e/bio

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 thefollowing formula.
Copy number of 1µg of plasmid DNA = 9.1 x 1011 / Size of plasmid DNA (kb)

[ 5 ] Protocol
1.

Standard reaction set up

Reaction volume

Final

Reagent
Sterilized water

50μL
X μL

20μL
X μL

Concentration

THUNDERBIRDTM Next 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(Uracil-N-glycosylase)[optional]

1 unit*2

0.4 units*2

DNA solution
Total

Y μL
50 μL

Y μL
20 μL

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 Heat-labile uracil-N-glycosylase (UNG) should be used. UNG is not supplied withthis kit. It is recommended that you use Uracil-DNA Glycosylase (UNG), Heat-labile(UNG-101) optionally.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

4

www.toyobo.co.jp/e/bio

2. Cycling condition
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] 2-4.
Fast cycling mode
<2-step cycle>

Time

Ramp

(UNG treatment)(20–25°C*1)(10 min*1)(Maximum)Pre-denaturation

95°C

30 sec*2

Maximum

95°C

5 sec*3

Maximum

Denaturation:

Temperature

40 cycles

60°C*410 sec*5
Maximum(data collection should be set at the extension step)
Melting / Dissociation Curve Analysis*6

Extension:Standard cycling mode
<2-step cycle>

Time

Ramp

(UNG treatment)(20–25°C*1)(10 min*1)(Maximum)Pre-denaturation

95°C

30 sec*2

Maximum

95°C

5 sec*3

Maximum

Denaturation:

Temperature

40 cycles

60°C*430 sec*5
Maximum(data collection should be set at the extension step)
Melting / Dissociation Curve Analysis*6

Extension:*1 [Optional] The indicated temperature and time are typical conditions for UNG. The

conditions can be optimized according to the particular instruction manual from the
supplier of UNG. Uracil-DNA Glycosylase (UNG), Heat-labile (Code No. UNG-101)
can be used.
*2 Due to the anti- DNA polymerase antibody hot start PCR system, the pre-denaturation

can be completed within 60 sec. The pre-denaturation time should be determinedaccording to the recommendations of each real-time cycler. If the optimal
pre-denaturation time cannot be determined, the time should be set at 30 sec.
*3 If the optimal denaturation time cannot be determined, the time should be set at 5 sec.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

5

www.toyobo.co.jp/e/bio*4 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.
*5 If the target size is smaller than 300 bp, the extension time can be set at 10 sec on

almost all real-time cyclers. Instability of the amplification curve or variation of datafrom each well may be improved by setting the extension time at 30-60 sec. Somereal-time cyclers or software need over 25 sec for the extension step. In these cases, thetime should be set according to each instruction manual (e.g. Applied Biosystems®
QuantStudio™ 5, 96-well, 0.2 mL: ≥ 25 sec; Applied Biosystems® 7000/73000: ≥ 31
sec; Applied Biosystems® 7500: ≥ 35 sec.).
*6 The melting curve analysis should be performed according to the recommendations ofeach real-time cycler.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

6

www.toyobo.co.jp/e/bio

2-1. Real-time PCR conditions using Applied Biosystems® StepOnePlus™
(Normal block type, software version 2.3)
The following is an example of a SYBR® Green I assay using Applied Biosystems®
StepOnePlus™. These conditions can also be useful for cyclers such as the Applied
Biosystems® 7500 Fast Real-Time PCR System and QuantStudio™ Real-Time PCR
System.

(1)

Select “Design Wizard”, “Advanced Setup” or “QuickStart” after starting thesoftware.

(2)

Select “SYBR® Green Reagents” as reagents in the following tabs.
Design Wizard
Advance Setup
QuickStart(3)

Methods & Materials
Setup  Experiment Properties
Experiment Properties

Select “Run Methods” and set the temperature conditions as follows:

*1
*3

*2

*1 Input of actual reaction volume is important to achieve a successful analysis.
*2 Set the data collection at the extension step. Some real-time cyclers or software needover 25 sec for the extension step. In these cases, the time should be set according toeach instruction manual (e.g. Applied Biosystems® QuantStudio™ 5, 96-well,
0.2mL:≥ 25 sec; Applied Biosystems® 7000/73000: ≥ 31 sec; Applied Biosystems® 7500:≥ 35 sec.).
*3 Add the condition for melting curve analysis.
(4) Insert the PCR tubes or plate.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

7

www.toyobo.co.jp/e/bio(5) Start the program.

2-2. LightCycler® 96(software version 1.1)
The following is an example of a SYBR® Green I assay using the Roche LightCycler®
96.

(1)

Select “Create New Experiment”.

(2)

Input reaction volume.

(3)

Select Preincubation  Preincubation  Two-Step Amplification 
Melting at “Predefined Program” in the “Run Editor” tab.

(4)

Select the first “Preincubation” and input 95 °C as “Target” and 30 s as “Duration”.

(5)

Select the second “2 Step Amplification” and input 95°C as “Target” and 5 s as
“Duration” followed by 60°C as “Target” and 10 s as “Duration”.

*1*2*1 Input of the actual reaction volume is important for a successful analysis.
*2 Set the data collection at the extension step.
(6) Insert the PCR tubes or plate.
(7) Start the program.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

8

www.toyobo.co.jp/e/bio

2-3. CFX96 Touch™ Deep Well
(software version 3.1)
The following is an example of a SYBR® Green I assay using the Bio-Rad
CFX96 Touch™ Deep Well.
(1)

Select “User-defined”.

(2)

Select “Create New …” and input reaction volume.

(3)

Input the following conditions: 95°C, 30 s, 95°C, 5 s, 60°C, 10 s, 40 cycles.

*1

*2
*3

*1 Input of the actual reaction volume is important for a successful analysis.
*2 Select “Insert Melt Curve” and add the cycle for the melting curve.
*3 Select “Add Plate Read to Step” and set the data collection point at the extension step.
(4)

Insert the PCR tubes or plate.

(5) Start the program.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

9

www.toyobo.co.jp/e/bio

2-4. 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 500 bp.
-PCR efficiency is low.

<3-step cycle>

Temperature

Time

Ramp

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:

40 cycles

10-60 sec*4

72°C
Maximum(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 Theannealing 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 (≤10 sec). However, certain
cyclers require >10 sec detection time at the extension step. An unstable signal may be
improved by prolonging the extension time up to 30-60 sec. Also note that somecyclers cannot have an extension time of 25 sec (e.g. Applied Biosystems®
QuantStudio™ 5, 96-well, 0.2 mL: ≥ 25 sec.; Applied Biosystems® 7000/73000: ≥ 31
sec.; Applied Biosystems® 7500: ≥ 35 sec.).

*4 Shorter targets (

*5

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

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

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

10

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

cDNA synthesis
cDNA synthesized by various cDNA synthesis reagents can be used with
THUNDERBIRDTM Next SYBR® qPCR Mix. However, cDNA synthesized by a reagent
specialized for real-time PCR can increase sensitivity.
ReverTra Ace™ qPCR RT Kit (Code No. FSQ-101), ReverTra Ace™ qPCR RT MasterMix (Code No. FSQ-201) and ReverTra Ace™ qPCR RT Master Mix with gDNA
Remover (Code No. FSQ-301) are cDNA synthesis kits suitable for real-time PCR.
Here, the protocol with ReverTra Ace™ qPCR RT Master Mix and ReverTra Ace™
qPCR RT Master Mix with gDNA Remover is described. However, for the detailed
protocol, please refer to the instruction manual of the kit.
ReverTra Ace™ qPCR RT Master Mix (Code No. FSQ-201)(1) Denaturation of RNA [optional]
Incubate the RNA solution at 65°C for 5 min, and then keep on ice.
Notes:-This step increases the efficiency of reverse transcription of RNA templates that form
secondary structures.
-This step should be performed before adding 5x RT Master Mix.
(2) Preparation of the reaction solution
Prepare the following reagents on ice.
5x RT Master Mix
RNA template

2 µL
1 pg – 1 µg

Nuclease-free Water

X µL

Total Volume

10 µL(3) Reverse transcription reaction-Incubate at 37°C for 15 min.
-Incubate at 50°C for 5 min. [optional]-Heat to 98°C for 5 min.
-Store the reacted solution* at 4°C or – 20°C
*This solution can be used in the real-time PCR reaction directly or after dilution.
Notes:-ReverTra Ace™ excels at high reaction temperatures (up to 50°C). This step may
increase the efficiency of the reverse transcription.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

11

www.toyobo.co.jp/e/bio

ReverTra Ace™ qPCR RT Master Mix with gDNA Remover (Code No. FSQ-301)(1) Preparation of the “4x DN Master Mix” and “gDNA Remover” mixture.
Prior to use, a 1 in 50 volume of gDNA remover should be added to 4x DN
Master Mix (e.g. 4x DN Master Mix : gDNA remover = 440 µL : 8.8 µL).
Notes
4x DN Master Mix with gDNA remover can be stored at -20°C for at least for 3 months. The mixture canbe prepared in a smaller volume [e.g. 4x DN Master Mix : gDNA remover = 220 µL : 4.4 µL].

(2) Denaturation of RNA [optional]
Incubate the RNA solution at 65°C for 5 min, and then keep on ice.
Notes
-This step increases the efficiency of reverse transcription of RNA templates that form
secondary structures.
-This step should be performed before adding 4x DN Master Mix.

(3) Preparation of the DNase I reaction solution:
Prepare the following reagents on ice.
2 µL

4x DN Master Mix
RNA template

0.5 pg – 0.5 µg

Nuclease-free Water

X µL

Total Volume

8 µL(4) Incubate at 37°C for 5 min.
(5) Preparation of the for reverse transcription solution;
Prepare the following reagents on ice.
Reacted solution from (4)8 µL

5x RT Master Mix II

2 µL

Total Volume

10 µL(6) Reverse transcription reaction-Incubate at 37°C for 15 min.
-Incubate at 50°C for 5 min. [optional]-Heat to 98°C for 5 min.
-Store the reacted solution* at 4°C or – 20°C
*This solution can be used in the real-time PCR reaction directly or after dilution.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

12

www.toyobo.co.jp/e/bio
Notes:-ReverTra Ace™ excels at high reaction temperatures (up to 50°C). This step may increase
the efficiency of the reverse transcription.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

13

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.

The template DNA is insufficient.

Loss 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 curves.

Competition with non-specificamplification.

The PCR efficiency islower than 80% (slope:<-3.95).

Inappropriate cycling conditions.
Degradation of the primers.
The calculation of the PCR
efficiency is inappropriate.
The calculation of the PCR
efficiency is inappropriate.

The PCR efficiency ishigher than 110%(slope: >-3.1).
Reproducibility is not
good.

Competition with primer dimer
formation.

Poor purification of the template
DNA
Absorption of the template DNA to
the tube wall.

Plasmid DNA or PCR product isused as a template.

Inappropriate thermal conditions.
Low purity of the primersJAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

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.

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

14

www.toyobo.co.jp/e/bio

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.
Detection of multiple
peaks on the melting
curve analysis

Inappropriate settings offluorescence measurement
Insufficient reaction volume.
Non-specific amplification.

Formation of primer dimer.

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

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 [5] (2). If the result is not
improved, the following should be performed:change the primer sequence and/or change thepurification 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.
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 [5] (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)

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

15

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

Code No.
QPS-101T

THUNDERBIRD™ Probe qPCR Mix

Package
1mL × 1(100 rxns)

QPS-101

High efficient cDNA synthesis kit for real-time PCR

1.67mL × 3(500 rxns)
200 rxns
200 rxns

FSQ-201

200 rxns

FSQ-301

0.5mL × 5(250 rxns)
0.5mL × 5(250 rxns)
250 rxns

QRT-101

200 U

UNG-101

High efficient real-time PCR master mix

FSQ-101

ReverTra Ace™ qPCR RT Kit
High efficient cDNA synthesis master mix for real-time PCR

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

ReverTra Ace™ qPCR RT Master Mix
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
One-step Real-time PCR master mix for probe assay

QRT-201
QRZ-101

THUNDERBIRD™ Probe One-step qRT-PCR Kit
Heat-labile Uracil-DNA Glycosylase

Uracil-DNA Glycosylase (UNG), Heat-labile

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

16