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F1236K

Instruction manual for KOD SYBR qPCR Mix 2004

KOD SYBR® qPCR Mix
QKD-201T
QKD-201

1 mL × 1
1.67 mL × 3

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

Introduction[2]

Components[3]

Primer design[4]Template DNA[5]

Protocol
1. Reaction mixture set up
2. PCR cycling conditions
2-1. Real-time PCR conditions using an Applied Biosystems StepOnePlus™
2-2. Real-time PCR conditions using a 7500 Fast Real Time System
2-3. Real-time PCR conditions using Roche LightCycler® Nano
2-4. Real-time PCR conditions using Bio-Rad MiniOpticon[6]

Quantitation using long or GC rich targets[7]

PCR fragment length polymorphism analysis using a melting curve assay[8]

SNP analysis by ASP-PCR[9]Related protocol
1. cDNA synthesis[10] Troubleshooting[11] Related products[12] References
CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe generallaboratory precautions and safety procedures 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, Inc.
.
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www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
KOD SYBR® qPCR Mix is a highly efficient 2× Master Mix for real-time PCR using
SYBR® Green I and based on the 3’-5’ exonuclease deficient KOD DNA Polymerase1)2)3).
The master mix contains all the required components, except the ROX reference dye andprimers (50× ROX reference dye is supplied separately with this kit). The master mix aids
reaction setup, and improves the reproducibility of experiments.
This product was optimized to be highly efficient and robust in the SYBR® Green assay.

Features-Effective for GC rich targets
Quantitative analysis can be achieved even at GC contents greater than 70%.
-Long target amplification (~2 kb)
Quantitative analysis can be achieved using long targets, up to 2kb. Therefore, primers forconventional PCR can be applied. This aids primer design.
This reagent enables PCR fragment length polymorphism analysis using a melting curve
assay.
-High specificity
Optimization and hot start technology permit the highly specific amplification.
-Effective amplification from crude samples
Effective amplification can beachieved using crude samples, as
shown in Table I. This reagent
can be used for genotyping or
SNP analysis using crude
specimens.

Table I Applicable crude samples

whole bloodnailhair rootoral mucosa
cultured cells
animal tissue
plant tissue

ca.1% (final)
ca 1mm
1~2 mmSuspension*~ 103 cells
lysate*lysate **See p 5-6-Compatibility with various real-time cyclers.
The reagent may be used in most real-time cyclers (i.e. Block type and glass capillary type).
The 50× ROX reference dye is individually supplied with this kit; therefore, the kit can beapplied to real-time cyclers that require a passive reference dye.

About the SYBR® Green I detection system
The SYBR® Green I assay system uses 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.

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

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

1 mL × 1
50 μL × 1

<QKD-201>
KOD SYBR® qPCR Mix
50× ROX reference dye

1.67 mL × 3
250 μL × 1

Notes:-KOD 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 protected from light. No
negative effect was detected by 10 freeze-thaw cycles of KOD SYBR® qPCR Mix. This
reagent does not contain the ROX reference dye.
-50× 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 1× or 0.1×. 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 3 shows the optimal concentrationof the ROX dye.
1× Solution
KOD SYBR® qPCR Mix: 50× ROX reference dye = 1.67 mL: 66.8 μL (QKD-201)
KOD SYBR® qPCR Mix: 50× ROX reference dye = 1 mL: 40 μL(QKD-201T)
0.1× Solution
KOD SYBR® qPCR Mix: 50× ROX reference dye = 1.67 mL: 6.7 μL
KOD SYBR® qPCR Mix: 50× ROX reference dye = 1 mL: 4 μL

(QKD-201)(QKD-201T)

For real-time cyclers that do not require a passive reference dye, KOD SYBR® qPCR Mix
can be used without the ROX reference dye.

[3]

Primer design

1. Primer conditions
Highly sensitive and quantitative data depend on good primer design. The primers should
be designed according to the following suggestions:-GC content of primer: 45-60%-Target length: ≤ 2 kp (optimal)-Melting temperature (Tm) of primers: 60-70°C-Purification grade of primers: Cartridge (OPC) grade or HPLC grade

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www.toyobo.co.jp/e/bio- Adding a G or C to the 3’ end of the primers can enhance priming efficiency.
-The following primers should not be used.
- 3’-ends that have complementarity.
-High GC content in the 3’ region.
-The primer has complementary regions.
-The Tm of primers should be calculated using the Nearest Neighbor method. The Tm
values in this manual were calculated using this method with the following parameters.
Na+ concentration: 50 mM
Oligonucleotide concentration: 0.5 µM[ 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 KOD 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 the cDNA
may inhibit the PCR. Up to 20% (v/v) of the cDNA solution from the 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.

2. Genomic DNA, Viral DNA
Genomic DNA and viral DNA can be used at up to 200 ng in 50 μL reactions.
Notes:The background tends to be high at the high copy range for genomic DNA, because thetemplate DNA intercalates SYBR® Green I.

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 using thefollowing formula.
Copy number of 1µg of plasmid DNA = 9.1 × 1011 / Size of plasmid DNA (kb)Linearity tends to be lost at the low copy range because diluted plasmid DNA is easily
adsorbed onto vessels. Linearity can be improved using solutions containing carriers (e.g.
yeast RNA) as diluents of template DNA.

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4. Crude samples
Crude samples can be used as templates. The samples should be added according to thefollowing guidelines, because excessive crude samples may inhibit the reaction anddetection.
Table 1
Applicable crude samples for 20 μL reaction
Whole blood
0.2μL (2μL: 1/10 diluted sample)
Nail (mouse toe)
1 mm
Hair
1–2 cm from a hair root
Oral mucosa
5 μL of 200μL suspension of oral mucosa collected bya cotton swab.
Cultured cells~ 103 cells
Animal tissue
0.5~2 μL of lysate (alkaline lysis method)
Plant tissue
0.5~2 μL of 1/10 diluted lysate (one step method)<Alkaline lysis method>
The following alkaline lysis method is recommended for rapid preparation of animal
tissue lysates (e.g. mouse tail lysate) suitable for amplification with KOD SYBR® qPCR
Mix.

*Animal tissue cannot be dissolved completely.

Table 2 Starting materials for preparing lysates
Mouse tail3 mm
Pig muscle
20 mg
Cow muscle
20 mg
Nail (mouse toe)
5 mg
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www.toyobo.co.jp/e/bio<One step method>
The following one-step method is recommended for the rapid preparation of a planttissue lysate suitable for amplification with KOD SYBR® qPCR Mix.

Notes:
Homogenization of plant tissue with a pestle in Buffer A enhances the efficiency. In thiscase, heating is not necessary.

[ 5 ] Protocol

1.

Reaction mixture setup

DW

Reaction volume
50μL
20μL
X μL
X μL

KOD SYBR® qPCR Mix

25 μL

10 μL

1×

Forward Primer

10 pmol

4 pmol

0.2 μM*1

Reverse Primer

10 pmol

4 pmol

0.2 μM*1

50X ROX reference dye

1μL / 0.1μL 0.4μL / 0.04μL

DNA solution

Y μL

Reagent

Total

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50 μL

Final
Concentration

1× / 0.1×*2

Y μL

20 μL

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Notes:*1 Higher primer concentration tends to improve amplification efficiency; lower primer
concentration tends to reduce non-specific amplification. The primer concentration
should be between 0.05-1.0 µM.
*2 50× ROX reference dye must be added when using real-time cyclers that require a
passive reference dye (i.e. Applied Biosystems, Agilent), according to Table 3. Table 3
shows the optimum concentration of the ROX reference dye. This dye is not necessary
for real-time cyclers that do not require a passive reference dye.
Table 3 Recommended ROX dye concentration
Real-time cycler
Applied Biosystems 7000, 7300, 7700, 7900HT
StepOne™, StepOnePlus™ etc.
Applied Biosystems 7500, 7500Fast,
Agilent Technologies cyclers (Option) etc.
Roche cyclers, Bio-Rad cyclers, BioFlux cyclers etc.

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

Notes:The pre-mixed reagents can be prepared according to the following ratios:
1× Solution
KOD SYBR® qPCR Mix: 50x ROX reference dye = 1.67 mL: 66.8 μL
KOD SYBR® qPCR Mix: 50x ROX reference dye = 1 mL: 40 μL(QKD-201)(QKD-201T)

0.1× Solution
KOD SYBR® qPCR Mix: 50x ROX reference dye = 1.67 mL: 6.7 μL
KOD SYBR® qPCR Mix: 50x ROX reference dye = 1 mL: 4 μL

(QKD-201)(QKD-201T)

2. PCR cycling conditions(A) Recommended cycling conditions.
The following table shows the recommended thermal conditions using primers designed
according to the recommendations described in [3]. Almost all targets can also be
amplified using the following conditions with other real-time PCR reagents.
If satisfactory results are not obtained, cycling condition should be altered according to
the next section (B).
<3-step cycle>
Pre-denaturation
Denaturation:
Annealing
Extension:(40 cycles) *4

Time
Ramp
*1
2 min.
Maximum
10 sec.
Maximum
10 sec.
Maximum
30 sec. / 500 bp*3
Maximum(Set 30 sec. in the case of ≤ 500 bp)<data collection should be set at the extension step>
Melting / Dissociation Curve Analysis*5
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Temperature
98°C
98°C
60°C*268°C

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www.toyobo.co.jp/e/bio*1 Pre-denaturation can be completed within 120 sec. because of the anti-KOD antibody hot
start PCR system.
*2 Insufficient amplification may be improved by decreasing the annealing temperature (toapprox. 50°C), and non-specific amplification (e.g. abnormal shapes of the amplification
curve at low template concentrations) may be reduced by increasing the annealing
temperature (to approx. 68°C).
*3 When the target size is smaller than 500 bp, the extension time can be set at 30 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 45–60 sec. Some realtime cyclers or software need over 30 sec. for the extension step. In these cases, the timeshould be set according to each instruction manual (e.g. Applied Biosystems 7000/7300:≥ 31 sec.; Applied Biosystems 7500: ≥ 35 sec.).
When the target size is larger than 500 bp, the extension time should be calculated as 30
sec. / 500 bp.
*4 When crude samples are used as templates, the Ct may be delayed. In such cases, thecycle number should be increased up to 50.
*5 Melting curve analysis is important to evaluate the specificity of the intercalation assay
using SYBR® Green I. In the case of targets having >80% GC content, the upper limitof the melting temperature should be set at 99°C. When the melting curve analysis isincomplete, specificity should be confirmed by electrophoretic analysis on an agarose
gel.
(B) Optimization of PCR cycling conditions
In cases of low specificity or poor amplification, the following optimization steps may beeffective:<Low specificity>
Higher annealing temperature or two-step cycling may improve the specificity. The
annealing temperature should be determined to achieve a balance of specificity andamplification efficiency.
(Standard conditions)
98°C, 10 sec. 
60°C, 10 sec.



68°C, 30 sec./500 bp (40 cycles)(Increase the annealing temperature)
98°C, 10 sec.  ~68°C, 10 sec. 

68°C, 30 sec./500 bp (40 cycles)(2 step cycling)
98°C, 10 sec. (40 cycles)





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68°C, 30 sec./500 bp

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www.toyobo.co.jp/e/bio<Poor amplification (no amplification)>
Elongation of the extension time or a lower annealing temperature may improve
amplification efficiency. Increasing the primer concentration may also improve efficiency.
(Standard conditions)
98°C, 10 sec. 
60°C, 10 sec.



68°C, 30 sec./500 bp (40 cycles)(Elongation of the extension time)
98°C, 10 sec. 
60°C, 10 sec.



68°C, ~ 1 min./500 bp (40 cycles)(Decreasing the annealing temperature*1)
98°C, 10 sec. 
50°C~, 10 sec. 
*1

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68°C, 30 sec./500 bp(40 cycles)

The following functions are convenient for optimization of the conditions:VeriFlex™: ABI StepOnePlus™, Thermal gradient: Bio-rad

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2-1.

Real-time PCR conditions using Applied Biosystems StepOnePlus™
(Normal block type, software version 2.2.2)

The following is an example of a SYBR® Green I assay using Real-time PCR conditions
using Applied Biosystems StepOnePlus™.

(1) Select “Design Wizard”, “Advanced Setup” or “QuickStart” after starting the software.
(2) Select “SYBR® Green Reagents” as reagents in the following tabs.
Design Wizard
Advance Setup
QuickStart

Methods＆Materials
Setup  Experiment Properties
Experiment Properties(3) Select “Run Methods” and set the temperature conditions as follows:*1 Input of actual reaction volume is important to achieve a successful analysis.
*2 Select “Add Step” and change the setting from a 2-step to a 3-step cycle.
*3 Set the data collection at the extension step.
*4 Add the condition for melting curve analysis. When the GC content of the target is high,the upper limit of the melting temperature can be set at 99°C.

(4) Insert the PCR tubes or plate(5) Start the program

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2-2. Real-time PCR conditions using Applied Biosystems 7500 Fast RealTime System(Normal block type, software version 1.4)
The following is an example of a SYBR® Green I assay using Real-time PCR conditions
using the Applied Biosystems 7500 Fast Real Time System.
(1) Select the “Instrument” tab after starting the software.
(2) Set the conditions as follows:*1 Input the correct reaction volume.
*2 Set the data collection at the extension step.
*3 Add the condition for melting curve analysis. When the GC content of the target is high,the upper limit of the melting temperature can be set at 99°C. When the GC content of
the target is >80%, the melting curve may be incomplete.
(3) Insert the PCR tubes or plate(4) Start the program

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2-3. Real-time PCR conditions using Roche LightCycler® Nano
(Software version 1.0)
The following is an example of a SYBR® Green I assay using the Roche LightCycler® Nano.
(1) Click “New” button, after starting the software.
(2) Input a title in the “Name” box in the “Experiment” tab.
(3) Select “Intercalating Dyes” in the “Run Settings” tab.
(4) Select “Profile” tab and set the cycling condition as follows:a. Click “add” and select “Hold”.
b. Alter the temperature conditions to 98°C, 120 sec, 5°C/sec.
c. Click “add” and select “3-Step Amplification”.
d. Set the temperature conditions as follows:e. Check box of “Acquire” at extension step.

f. Click “Add” and select “Melting”.
g. Confirm the settings as follows:(5) Insert the PCR tubes(6) Start the program

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2-4. Real-time PCR conditions using Bio-Rad MiniOpticon(Software version 2.0)
The following is an example of a SYBR® Green I assay using the Bio-Rad MiniOpticon(1) Select “Create a new run”, after starting the software.
(2) Select “Create New…” and set the temperature conditions as follows:*1 Input correct reaction volume.
*2 Select “Insert Step” and change the setting from “2-Step” to “3-Step”.
*3 Select “Insert Melt Curve” and add the cycle for the melting curve. When the GC content
of the target is high, the upper limit of the melting temperature can be set at 99°C.
*4 Select “Add Plate Read to Step” and set the data collection point at the extension step.

(3) Insert the PCR tubes or plate(4) Start the program

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www.toyobo.co.jp/e/bio[ 6 ] Quantitation using long / GC rich targets
Application data 1: Long target amplification<Method>
The efficiency was compared between KOD SYBR® qPCR Mix and conventional taq-based
qPCR Master Mix for the amplification of the human TGFβ gene (1.9 kb) from humangenomic DNA (10n dilution) and no-template control (NTC).
PCR cycling condition of KOD SYBR® qPCR Mix:
(ABI StepOnePlus™)<3-step cycle>
Temperature
Time
Ramp
Pre-denaturation
98°C
2 min.
Maximum
Denaturation:
98°C
10 sec.
Maximum
Annealing
60°C
10 sec.
Maximum
Extension:
68°C
2 min.*
Maximum(40 cycles)<Data collection was set at the extension step>
Melting / Dissociation Curve Analysis*The extension time was set 30 sec/500bp.
Target: TGFβ (1.9 kb)<Result>
KOD SYBR® qPCR Mix amplified the target (1.9 kb) successfully and quantitatively,
whereas the conventional Taq-based master mix (THUNDERBIRDTM SYBR® qPCR Mix)
showed poor amplification.

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Application data 2: Amplification of a GC-rich target<Method>
The amplification efficiency was compared between KOD SYBR® qPCR Mix and highefficiency qPCR master mix compatible with high GC targets. Diluted cDNA (5n)synthesized from HeLa cell total RNA using ReverTra AceTM qPCR RT Kit (Code: FSQ101) was used as the template.
PCR cycling condition of KOD SYBR® qPCR Mix:
(ABI StepOnePlus™)<3-step cycle>
Temperature
Time
Ramp
Pre-denaturation
98°C
2 min.
Maximum
Denaturation:
98°C
10 sec.
Maximum
Annealing
60°C
10 sec.
Maximum
Extension:
68°C
30 sec.
Maximum(40 cycles)<Data collection was set at the extension step>
Melting / Dissociation Curve Analysis
Target: IGF2R gene (189 bp, GC content: 83%)
<Result>
KOD SYBR® qPCR Mix amplified the high GC target successfully and quantitatively,
whereas the high efficiency qPCR master mix compatible with high GC targets (CompanyA) showed poor amplification and generated primer dimers.

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www.toyobo.co.jp/e/bio[ 7 ] PCR fragment length polymorphism analysis using a melting curve assay
KOD SYBR® qPCR Mix aids the design of amplicons having various Tm values becauseof its amplification ability up to 2 kb. Therefore, the reagent is able to realize multiplex
PCR or a PCR fragment length polymorphism assay in one tube using melting curve
analysis. In this analysis, the difference of Tm between the fragments should be > 3°C(optimally >5°C).
<Method>(a)(b)

Temperature (°C)(c)
Fragment
(bp)
60
94
141
199
357
552
1,061

Tm (°C)*1<Predicted>
72
76
79
82
84
86
88

Tm (°C)<Measured>
74
77.5
79
82
85
86
87

Amplification of β-actin gene with various
primers.
(a) Agarose gel electrophoresis analysis ofamplicons.
(b) Melting curve analysis of amplicons.
(c) Comparison of the calculated and measured
melting temperatures (Tm).

*1: The melting temperature (Tm) was calculated by the following formula.
Tm = 64.9 + 41×(yG+zC-16.4) / (wA+xT+yG+zG)(W, x, y and z represent the number of A, T, G and C nucleotides in a fragment,
respectively.)
Wallace RB et al. (1979) Nucleic Acids Res 6:3543-3557
Sambrook, J., and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual.

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16www.toyobo.co.jp/e/bio<Construction of primers for genotyping using one common and two specific primers>
The following is a strategy for constructing primers for genotyping with PCR fragment
length polymorphism using the KOD SYBR® qPCR Mix. This method is based on the
difference in the melting temperatures between amplicons.
(1) Design a primer for a small fragment
Primers should be designed such that the amplicon size is between 70 and 150 bp. Although
the short target is set on the wild-type (WT) in the following example, the target can be set
on both alleles.
Common primer
Primer S
Size: 70bp~150bp
Wild-type (WT)Knock-in

Knock in (KI)(2) Calculate the Tm of the small fragment (refer to the formula on the previous page).
(3) Design a primer for a large fragment
Primers for large fragments (Primer L) should be designed such that the difference of Tm
between small and large targets is greater than 3°C (optimally 5°C). The size of the longtarget should be less than 500 bp (optimally 300 bp) to prevent decreased PCR efficiency.
If the difference of Tm is less than 3°C, the primer for the small fragment should beredesigned so that the difference of Tm is larger than 3°C.
Common primer

Primer S

WT∆: >3°C (optimally > 5°C)

Knock-inKI

Primer L(4) Preliminary experiment
The preliminary experiment should be done using the primers along with the above
suggestions and appropriate templates (in this experiment, a heterozygous template isuseful). The ratio of primer should be determined based on the following table, because thesignal intensity is proportional to the amount (size) of the amplicon, not number of molesof it in SYBR® Green I assay.

Primer L
Common primer
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Primer concentration
0.2μM x (small fragment [bp]) / (large fragment [bp])
0.2μM

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www.toyobo.co.jp/e/bio(5) Adjustment of peak intensities
When the intensities of the peaks are different, the concentration of the primer for the larger
peak should be reduced. In the following experiment, the primer for the large fragment(Primer L) was changed. In this experiment, decreasing the concentration of Primer L
increased the peak of the small fragment.
Common: S : L =3: 3: 1.5

Temperature (°C)

3:3:1

3 : 3 : 0.75 (Molar ratio)

Temperature (°C)

Decreasing the peak of the small fragment

Temperature (°C)
Increasing the peak of the small fragment(Small fragment: 100 bp, Large fragment: 341 bp)

Application data 3:

Genotyping using mouse-tail lysates

Genotyping was performed using mouse-tail lysates prepared by the alkaline lysis method.
In this experiment, 100 bp (predicted Tm: 79°C) and 341 bp (predicted Tm: 84°C)
fragments were amplified and analyzed. The primers were designed such that the differencein Tm was greater than 5°C.
Mouse-tail (ca.3mm)| 50mM NaOH 180 μL| Vortex| 95°C, 10 min.
| 1M Tris-HCl (pH8.0) 20 μL| Vortex| 12,000rpm, 5 min. (optional)
Supernatant
0.5 ~ 2 μL

20 μL (reaction)(ABI 7500 Fast Real Time System)<3-step cycle>
Temperature
Time
Ramp
Pre-denaturation
98°C
2 min.
Maximum
Denaturation:
98°C
10 sec.
Maximum
Annealing
60°C
10 sec.
Maximum
Extension:
68°C
30 sec.
Maximum*1(40 cycles)<data collection was set at the extension step>
Melting / Dissociation Curve Analysis*1 Minimal cycling number should be chosen to prevent primer dimer generation.
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www.toyobo.co.jp/e/bio[ 8 ] SNP analysis by ASP-PCR
The melting temperature of a PCR product can be increased by 3~5°C by adding a GC tailat the 5’ end. This method uses one tube ASP (Allele specific primer)-PCR for SNP (Single
nucleotide polymorphism) analysis. The primer design is the same as for ordinary ASPPCR except for GC tailing. A SNP site should be located at the 3’ region and is analyzed
based on with or without amplification.
<Primer design>-The target should be less than 100 bp to permit different Tms between amplicons.
-This method can be applied to various kinds of ASP-PCR. In Application data 4, SNP andmismatch sites were set at the second and third bases from the 3’ end of the primer,
respectively. In this case, the bases at the mismatch sites can be any bases except T, whichcorresponds to A. In this case, G and A were used.
-The difference in Tms between amplicons should be greater than 3°C*1. If the differencein the Tms is less than 3°C, the amplicon size should be decreased.
-The sequence of the GC tail can be flexible. Various kinds of GC tails have been reported.
The following figure is an example of constructing primers for SNP analysis of ALDH2.
-If the intensities of the peaks are different, the primer concentration should be reconsideredaccording to the previous section “Construction of primers for genotyping using one
common and two specific primers”.
Allele specific primer

5’

CommonPrimer
3’
5’

ACG
TGA

SNP siteCommonAmplicon with a GC tail3’

Mismatch site∆: >3°C *1

Allele specific primer

Primer
3’
5’

ATA
TAA

SNP site

5’
3’

Amplicon without GC tailMismatch site

*1 The “Smoothing function” sometimes generates one broad peak from two adjacent peakson the melting curve analysis when using several real-time cyclers. In such a case, thedifference in Tms between two amplicons should be greater than 5°C, achieved bydecreasing the amplicon size in decrements up to 50~60bp.

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Application data 4:

Detection of ALDH2 SNP from whole blood and oral mucosa<Method>
SNP of ALDH2 (aldehyde dehydrogenase 2) were detected directly from specimens ofwhole blood and oral mucosa, without purification. On this analysis, a primer with a GC
tail was used to amplify the wild-type allele to make a difference in Tm between theamplicons.

Common primer

GC tail

5’

3’G
Common primer

5’

3’
A

(Sequences around the SNP site of ALDH2
487Glu5’------ ctgcaggcatacact GAA gtgaaaactgtga ------3’
487Lys5’------ ctgcaggcatacact AAA gtgaaaactgtga ------3’(Primers for ASP-PCR analysis)
Common primer: 5'-GTACGGGCTGCAGGCATAC-3'
G specific primer: 5'-GCCGCCCTGCCCGCCACACTCACAGTTTTCACTGCA-3'
A specific primer: 5'-CCCACACTCACAGTTTTCACTATA-3': Mismatch site; : SNP site
(Amplification size and Tm)
TargetCommon primer – G specific primer
Common primer – A specific primer

Size
57 bp
45 bp

Tm (Predicted)
78°C
72°C

Template DNAs was prepared by the following methods and added to the reaction mixtures
directly.

(ABI 7500 Fast Real Time System)<3-step cycle>
Temperature
Time
Ramp
Pre-denaturation
98°C
2 min.
Maximum
Denaturation:
98°C
10 sec.
Maximum
Annealing
60°C
10 sec.
Maximum
Extension:
68°C
30 sec.
Maximum(50 cycles)<Data collection was set at the extension step>
Melting / Dissociation Curve Analysis

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www.toyobo.co.jp/e/bio(Pretreatment of the specimens)
Whole blood 2 μL
Suspend in 48 μL DW
Suspension 5 μL

20 μL (reaction)

Cotton swab with oral mucosa
Suspend in 200 μL DW
Suspension 5 μL

20 μL (reaction)<Result>

Temperature (°C)

Temperature (°C)

SNP typing was achieved successfully without purification of templates.

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www.toyobo.co.jp/e/bio[ 9 ] Related protocol

1. cDNA synthesis
cDNA synthesized by various cDNA synthesis reagents can be used with KOD 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. However, this step may increase the efficiency of the reverse
transcription of RNA, which forms secondary structures.
-Do not add 5× RT Buffer and/or enzyme solution at this step.
(2) Preparation of the reaction solution

Reagent
Nuclease-free Water
5× 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 5 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[ 10 ] Troubleshooting
Symptom

Loss of linearity in thehigh cDNA/DNA
concentration region.

Cause

SolutionIntercalation of SYBR® Green I into
the template DNA.

SYBR® Green I is also intercalated into thetemplate DNA; therefore, the baseline tends to behigher when high concentration DNA samples areused. Diluted template should be used to obtain acorrect Ct value.
Excessive crude specimen inhibits the PCR
reaction. The amount of specimen should bedecreased or the specimen should be purified.
cDNA synthesis reagents designed for real-time
PCR should be used.

Inhibition of the reaction by PCR
inhibitors in crude samples
cDNA synthesis reagent is
inappropriate (a certain component
inhibits the reaction)
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 carves.

Competition with non-specificamplification.

Competition with primer dimer
formation.

Circular plasmid DNA
The PCR efficiency islower than 80% (slope:<-3.95)

The PCR efficiency ishigher than 110%(slope > -3.1)

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Inappropriate cycling conditions.
Tm of primers are low

Degradation of the primers.
The calculation of the PCR
efficiency is inappropriate.
Primer concentration is low
The calculation of the PCR
efficiency is inappropriate.
Non-specific amplification

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
before the experiments.
Dimer formation may reduce the amplification
efficiency of the target, especially for reactions at
low template concentrations. 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.
Linearized plasmid DNA should be used because
circular DNA tends to cause data spread.
Optimize the cycling conditions according to [5]2. PCR cycling conditions.
Annealing is insufficient. Optimize the cycling
conditions according to [5] 2. PCR cycling
conditions.
Fresh primer solution should be prepared.
The Ct value on the linear region should be used
to calculate the PCR efficiency.
Increase the primer concentration.
The Ct value on the linear region should be used
to calculate PCR efficiency.
Non-specific amplification may raise the apparent
PCR efficiency over 110 %. The specificity
should be confirmed by melting curve analysis.

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Symptom

Cause

Solution

Reproducibility is not
good.

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

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 before theexperiments.
In general, plasmid DNA or PCR products areused at low concentrations. Diluted DNA
templates tend to be absorbed onto the tube wall.
Dilution of the template DNA/cDNA should be
performed before the experiments. Dilution with acarrier nucleic acid solution (Yeast RNA) is also
effective in improving linearity.
Different batches of primers may generate
different results. When the batch is changed, priortesting of the primer should be performed.
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. PCR cycling
conditions. If the result is not improved, thefollowing should be performed: change the primer
sequence and/or change 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. 50× ROX reference dye should
be used according to [5] Table 3.
Settings should be confirmed according to the
instruction manual of each detector. Prolonged
extension time (45~60 sec.) may improve theinstability of the amplification curve.
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. PCR cycling
conditions. If the result is not improved, thefollowing action should be performed: change theprimer sequence and/or change the purification
grade of the primer(HPLC grade)

Plasmid DNA or PCR product isused as a template.

Low purity of the primers

Amplification from thenon-template control(NTC).

Formation of primer dimers.

Contamination or carry over of the
PCR products.

Low amplification
curve signal /Unstable amplification
curve signal.

Excessive amount of ROX referencedye.
Inappropriate settings offluorescence measurement

Insufficient reaction volume.
Detection of multiple
peaks on the melting
curve analysis

Non-specific amplification.

Formation of primer dimer.

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Symptom
GC tail does notincrease the Tm of
amplicons

Cause

SolutionTarget size is too large

GC content of the target is too highPeaks from multiplex
PCR
cannotbeseparated on melting
curve analysis

Target size is too large

GC content of target is too highEffect of “Smoothing function” of areal-time cycler.

Intensities of peaks
from multiplex PCR
are different on
melting curve analysis

Sizes of the targets are too different

Annealing efficiency of the primers
are different

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The effect of the GC tail will be low when theamplicon size is long. Shorten the amplicon size
to below 100 bp. Tms of DNA fragments can becalculated using the algorithm in [7] PCR
fragment length polymorphism.
When the GC content of the target is high, theeffect of the GC tail will be low. Targets andprimers should be determined in consideration ofTm.
Tms of targets can be increased by enlarging theirsizes, to some extent (~ 1 kb). The fragment size
should be determined in consideration of thedifference in Tm of each PCR product using thealgorithm in [7] PCR fragment length
polymorphism.
When the GC content of the target is high, theeffect of GC tail will be low. Targets and primers
should be determined in consideration of Tm.
“Smoothing function” sometimes generates one
broad peak from two adjacent peaks on themelting curve analysis when using several realtime cyclers. In such cases, the difference in Tms
between two amplicons should be greater than
5°C, achieved by decreasing the amplicon size indecrements up to 50~60bp.
A signal intensity is in proportion to the amount(size) of an amplicon, not the number of moles of
it in the SYBR® Green I assay. Primer
concentration should be determined according to[7] PCR fragment length polymorphism.
The difference in Tms of primers may generatepeak imbalances. In such cases, the primer
concentration for the higher peak should bedecreased according to [7] PCR fragment length
polymorphism.

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www.toyobo.co.jp/e/bio[ 11 ] Related products

Product name

Package
1mL × 11.67mL × 3
1mL × 11.67mL × 3
200 rxns

Code No.
QPS-101T
QPS-101
QPS-201T
QPS-201
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 × 5

QRT-101

0.5 mL × 5

QRT-201

High efficient real-time PCR master mix for probe assay
THUNDERBIRDTM Probe qPCR Mix
High efficient real-time PCR master mix for SYBR® Green assay
THUNDERBIRDTM SYBR® 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[ 12 ] References
1)

Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M,
and Imanaka T., Appl Environ Microbiol., 63: 4504-10 (1997)

2)

Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y, J
Mol Biol., 306: 469-77 (2001)

3)

Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126:
762-8 (1999)JAPANTOYOBO CO., LTD.
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