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F0924K

Instruction manual SYBR® Green Realtime PCR Master Mix 2004

SYBR® Green Realtime PCR Master Mix
QPK-201

1 mL x 5

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

Introduction[2]

Components[3]

Primer design[4]Detection[5]

Specimens[6]

Protocol
1. Intercalation assay protocol using ABI PRISM® 7700
2. Intercalation assay protocol using Roche LightCycler™
3. One-step RT-PCR using reverse transcriptase[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.
-LightCycler™ is a trademark of Idaho Technology, Inc. and Roche Molecular Systems, Inc.
-SYBR® is a registered trademark of Molecular Probes, Inc.
-ABI PRISM® is a registered trademark of Perkin-Elmer Corporation.
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1www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
This product is a Taq DNA polymerase-based 2 x master mix for real-time PCR, whichcontains all components, except for the primer. This reagent is applicable for intercalation
assay with SYBR® Green I.

Features-This reagent can be used in glass capillary systems (e.g., LightCycler, Roche MolecularSystems, Inc.).
-This reagent can be used in a passive reference system (e.g., ABI PRISM® 7700, AppliedBiosystems, Inc.). The passive reference dye does not affect any other systems.
-Hot Start technology with anti-Taq DNA polymerase antibodies enables high specificity
and reproducible amplification.

[ 2 ] Components

This reagent includes the following components for 200 reactions (QPK-201), 50 µl total
reaction volume:<QPK-201>
SYBR® Green Realtime PCR Master Mix

1 mLx 5Notes:
This reagent can be stored at 4°C for 2 months and protected from light. For longer storage,
this reagent should be kept at -20°C and protected from light.

[3]

Primer design
Primers should be designed according to the following guidelines:-Primer length: 20~30 mer-GC content of primer: 40~60%-Target length: ≤ 200 bp (optimally, ≤ 150bp)
Notes:-Longer targets (>200 bp) reduce efficiency and specificity of amplification. The idealtarget length range is 50~150 bp.
-Since detection in an intercalation assay is affected by non-specific amplification, primers
should be as specific as possible.

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www.toyobo.co.jp/e/bio[ 4 ] Detection

This reagent can be used in general detection devices, such as:-LineGene (Bioer Technology co., ltd.)
This reagent can also be used in detection equipment using glass capillaries or passivereference, such as:
LightCycler (Roche Molecular Systems)
ABI PRISM® 7000, 7700, and 7900 (Applied Biosystems)
Note:

The passive reference mode of detectors should be set at “ROX”.

Fig. 1[ 5 ] Specimen

Amplification curves (left) and melting curves (right) with SYBR® Green Realtime PCR
Master Mix

1. cDNA-Reverse transcription reactions from total or poly (A)+ RNA may be used directly, orafter dilution, for real-time PCR. Purified cDNA by phenol/chloroform extraction andethanol precipitation may also be used. Oligo dT and random primers are suitable forthe reverse transcription reaction.
-Up to 20% of the synthesized cDNA solution from the ReverTra AceTM qPCR RT Kit(Code NO. FSQ-101) may be added to the PCR reaction solution directly, withoutpurification.

2. Genomic DNA-Purified DNA, which would be used for general PCR, is also suitable for real-time PCR.
In the case of mammalian genomic DNA, 1~10 ng genomic DNA is sufficient for realtime PCR.

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

1.

Intercalation assay protocol using ABI PRISM® 7700
The following is an intercalator assay protocol to be used with ABI PRISM® 7700. Forother detection devices, this protocol may require modification depending on each
instruction manual.
(1) Preparation of reaction solutionComponent
PCR grade water
SYBR® Green Realtime PCR Master Mix
10pmol/μL(10 µM) Primer #1
10pmol/μL (10 µM) Primer #2
Template DNA
Total volume

Volume
16 μL
25 μL
2 μL
2 μL
5 μL
50 μL

Final Concentration
1x0.4 µM
0.4 µM

Notes-The primer concentration can be further optimized, if needed. The optimal range for theprimers is 0.2~0.6 µM. In the case of commercially available primers, recommended
conditions from those companies should be used.
(2) Cycling condition

The following condition is recommended. Initially, a 3-step cycle should beused.
<3-step cycle>
Pre-denaturation:

95°C, 1 min.

Denaturation:

95°C, 15 sec.

Annealing:

55~65°C, 15 sec.

Extension:

72°C, 45 sec. (data collection)

40 cycles

Melting curve analysis<2-step cycle>
Pre-denaturation:

95°C, 1 min.

Denaturation:

95°C, 15 sec.

Extension:

60°C, 1 min. (data collection)

40 cycles

Melting curve analysis
Notes-The annealing temperature in 3-step cycle should be set to 55~65°C, depending of theprimer Tm value.
-The pre-denaturation condition described above is sufficient for inactivation of the antiTaq DNA polymerase antibodies used in Hot Start PCR. To prevent unexpected andinappropriate results, do not prolong the pre-denaturation period. Fifteen seconds isalso sufficient for denaturation during each cycle.
-Data collection step should be longer than 30 sec.
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www.toyobo.co.jp/e/bio

2. Intercalation assay protocol using Roche LightCycler™
The following is an intercalator assay protocol to be used with the Roche LightCycler™.
In the case of other detection devices, this protocol should be modified accordingly.
(1) Preparation of reaction solution
Component

VolumePCR grade water
SYBR® Green Realtime PCR Master Mix
10pmol/μL (10 µM) Primer #1
10pmol/μL (10 µM) Primer #2
Template DNATotal volume

6.4 μL10 μL
0.8 μL0.8 μL
2 μL
20 μL

FinalConcentration
1x0.4 µM
0.4 µM

Notes-The primer concentration can be further optimized, if needed. The optimal range forprimers is 0.2~0.6 µM. In the case of commercially available primers, recommended
conditions from each manual should be followed.
(2) Cycling conditions

The following condition is recommended:<3-step cycle>
Pre-denaturation:

95°C, 30 sec.

Denaturation:

95°C, 5 sec.

Annealing:

55~65°C, 10 sec.

Extension:

72°C, 15 sec. (data collection)

40 cycles

Melting curve analysis
Notes-The annealing temperature can be set to 55~65°C, depending on the primer Tm value.
-The annealing time should be set for 5~20 seconds. Longer annealing time results inincreased efficiency, and a shorter time decreases non-specific amplification.
-The pre-denaturation condition described above is sufficient for inactivation of the antiTaq DNA polymerase antibodies used in Hot Start PCR. To prevent unexpected andinappropriate results, do not prolong the pre-denaturation period. Five seconds is alsosufficient for denaturation during each cycle.
-Data collection step should be longer than 10 sec.
-If commercially available primers or probes are employed, the recommended
conditions from each company should be used.

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3.

One-step RT-PCR by adding a reverse transcriptase
This reagent can be used for a one-step intercalator assay using reverse transcriptase.
The following is a one-step protocol using this reagent and a high-efficiency reverse
transcriptase, “ReverTra AceTM (Code No. TRT-101)”.

(1) Preparation of diluted RTase solution. <This solution is used in (2)>Component

VolumeFinal
Concentration

PCR grade water
RNase inhibitor (40 U/μL) <Code No. SIN-201>
ReverTra AceTM (100 U/μL) <Code No.TRT-101>
Total volume

28 μL
4 μL
1 μL
33 μL

5 U/μL
3 UμL

Component

VolumeFinal
Concentration

PCR grade waterRealtime PCR Master Mix
10 pmol/μL(10 µM) Primer #1
10 pmol/μL(10 µM) Primer #2
Diluted RTase solution (ReverTra AceTM 3U/μL)
Template RNATotal volume11 μL
25 μL
2 μL
2 μL
5 μL
5 μL
50 μL

(2) Preparation of reaction solution

1x
0.4 µM
0.4 µM
0.3 U/ μL

Notes-Because this method tends to result in non-specific amplifications, the conditions
should be optimized. Primer and probe concentrations can be further optimized, ifneeded. In the case of commercially available primers or probes, recommended
conditions from each manual should be employed.
-The reverse transcriptase concentration should be decreased when primer dimmers aregenerated.

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www.toyobo.co.jp/e/bio(2) Cycling conditions for ABI PRISM® 7700.

The following cycle condition is recommended. In the case of other detection
devices, this protocol should be modified accordingly.
<3-step cycle>
Reverse transcription

42°C, 20 min.

Pre-denaturation:

95°C, 5 min.

Denaturation:

95°C, 15 sec.

Annealing:

55~65°C, 15 sec.

Extension:

72°C, 45sec. (data collection)

40 cycles

Melting curve analysis
Notes-The reverse transcription step should take place at 42°C for 20 minutes. The predenaturation step should be at 95°C for 5 minutes to inactivate the antibodies against Taq
DNA polymerase and reverse transcriptase.
-The annealing temperature can be set to 55~65°C, depending on the primer Tm value.

(3) Cycling conditions for Roche LightCycler™
The following cycle condition is recommended:<3-step cycle>
Reverse transcription

42°C, 20 min.

Pre-denaturation:

95°C, 5 min.

Denaturation:

95°C, 5 sec.

Annealing:

55~65°C, 10 sec.

Extension:

72°C, 15 sec. (data collection)

40 cycles

Melting curve analysis

Notes-The reverse transcription step should take place at 42°C for 20 minutes. The predenaturation step should be at 95°C for 5 minutes to inactivate the antibodies against Taq
DNA polymerase and reverse transcriptase.
-The annealing temperature can be set to 55~65°C, depending on the primer Tm value.

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www.toyobo.co.jp/e/bio[ 7 ] Troubleshooting
Symptom

Cause

Solution

Incorrect mode setting of detector
for the fluorescence dye.
Incorrect setting for data collection.

Confirm the detector setting.

Incorrect setting for sample position.

Reposition the sample tubes.

Inappropriate concentration ofprimers.

Optimize the concentration of primers according
to instructions (see [6]).

Inappropriate design of primers.

Confirm specificity and Tm value of the primers.

Inappropriate cycle conditions.

Low purity or quality of samples

Confirm the primer Tm value. A lower annealing
temperature increases amplification efficiency.
For GC rich targets, an elongated denaturation
time might be effective.
Check the purity or quality of sample DNA.

Failure or malfunction of the device

Check the device.

Low quality sample DNA.

Inappropriate concentration ofprimers.

Repurify the sample DNA by phenol/chloroform
extraction and ethanol precipitation, or othermethod.
Optimize the concentration of primers according
to instructions (see [6]).

Inappropriate design of primers.

Confirm specificity and Tm values of the primers.

Inappropriate cycle conditions.

Confirm the Tm values of the primers.

Variation of dispensed volume.

Increase the reaction volume.

Contamination ofamplicons or sample DNAs.

Use fresh PCR grade water, and remake theprimer solution and master mix.

Detection of non-specificamplification.

Optimize the primer and cycle conditions.

Confirm the data collection setting.

No amplification

Variation in detection

Signals in blankreactionsJAPANTOYOBO CO., LTD.
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www.toyobo.co.jp/e/bio[ 8 ] Related products
Product name

Package200
reactions
10,000 U

Code No.
FSQ-1012,500 U
1 mL x 5

SIN-201
QPK-101

Real-time PCR master mix for SYBR® Green assay (improved version)
SYBR® Green Realtime PCR Master Mix –Plus-

1 mL x 5

QPK-212One-step Real-time PCR master mix for probe assay

0.5 mL x 5

QRT-101

0.5 mL x 5

QRT-201

High efficient cDNA synthesis kit for real-time PCR
ReverTra AceTM qPCR RT Kit
High efficient cDNA reverse transcriptase
ReverTra AceTM

RNase inhibitor (Recombinant type)
Real-time PCR master mix for probe assay

TRT-101

Realtime PCR Master Mix

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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Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

8