Description
Product description
2×Universal qPCR Fluore Green Master Mix (No ROX adjustment) is a pre-mixed solution for 2× real-time quantitative PCR amplification, featuring high sensitivity and specificity. It is blue in color and serves as a loading tracer. The core component, Taq DNA polymerase, is thermally activated by an antibody method, effectively suppressing non-specific amplification caused by primer annealing during sample preparation. Additionally, it contains factors that enhance PCR amplification efficiency and promote the amplification of genes with different GC contents (30-70%), ensuring a good linear relationship within a broad quantitative range.
This product contains a special ROX Passive Reference Dye and is suitable for all qPCR instruments without the need for ROX adjustment based on different instruments.
Specifications
|
Catalog Number
|
N132031E
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N132031S
|
N132031M
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N132031L
|
|
Specifications
|
50 T（20 μL/rxn）
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250 T（20 μL/rxn）
|
2500 T（20 μL/rxn）
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5000 T（20 μL/rxn）
|
Storage
Ice bag transportation. Store at -20℃ in the dark. Valid for 18 months.
This product should avoid repeated freezing and thawing. The product contains the fluorescent dye SYBR Green I. When storing or preparing the reaction system, strong light exposure should be avoided.
Notes
1. It is recommended to use Arcegen One-Step cDNA Synthesis RT Premix (Cat#N132061) to effectively remove residual genomic DNA from RNA samples.
2. After thawing, Master Mix may present flocculent or white precipitates. Hold it in your hand to slowly dissolve it and gently invert it up and down to mix until the solution becomes clear. This does not affect the performance of the reagent.
3. For your safety and health, please wear a lab coat and disposable gloves when operating.
4. This product is for research use only.
Instructions
1. Reaction System
|
Component
|
volume（μL）
|
volume（μL）
|
Final concentration
|
|
2×qPCR Master Mix
|
25
|
10
|
1×
|
|
Forward Primer (10 μM)
|
1
|
0.4
|
0.2 μM
|
|
Reverse Primer (10 μM)
|
1
|
0.4
|
0.2 μM
|
|
Template DNA
|
X
|
X
|
-
|
|
Sterile ultrapure water
|
to 50
|
to 20
|
-
|
【Note】Before use, make sure to mix thoroughly to avoid vigorous shaking that may produce excessive bubbles.
1) Primer concentration: The typical final concentration of primers is 0.2 μM, but it can also be adjusted between 0.1-1.0 μM depending on the situation.
2) Template concentration: If the template is undiluted cDNA stock solution, the volume used should not exceed 1/10 of the total qPCR reaction volume.
3) Template dilution: It is recommended to dilute the cDNA stock solution 5-10 times. The optimal amount of template added should result in Ct values between 20-30 cycles for amplification.
4) Reaction volume: A reaction volume of 20 μL or 50 μL is recommended to ensure the effectiveness and reproducibility of target gene amplification.
5) Reaction set up: Please prepare the reaction mixture inside a laminar flow cabinet and use nuclease-free pipette tips and reaction tubes. Filter-tipped pipette tips are recommended. Avoid cross-contamination and aerosol contamination.
Standard Procedure
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Recycling Procedure
|
Temperature
|
Time
|
Cycle Number
|
|
Pre-denaturation
|
95℃
|
2 min
|
1
|
|
Denaturation
|
95℃
|
10 sec
|
40
|
|
Annealing/extension
|
60℃
|
30 sec★
|
|
Melting Curve Analysis
|
Default Settings
|
1
|
Fast Program
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Recycling Procedure
|
Temperature
|
Time
|
Cycle Number
|
|
Pre-denaturation
|
95℃
|
30 sec
|
1
|
|
Denaturation
|
95℃
|
3 sec
|
40
|
|
Annealing/extension
|
60℃
|
20 sec★
|
|
Melting Curve Analysis
|
Default Settings
|
1
|
【Note】The fast program is applicable to the vast majority of genes. For genes with complex secondary structures, the standard program can be attempted.
1) Annealing temperature and time: Please adjust according to the length of the primers and the target gene.
2) Fluorescence signal acquisition (★): Do not forget to enable fluorescence signal acquisition. Set up the experimental procedure according to the user manual of the instrument. The time settings for several common instruments are as follows:
20 sec: Applied Biosystems 7700, 7900HT, 7500 Fast
31 sec: Applied Biosystems 7300
32 sec: Applied Biosystems 7500
3) Melting curve: The default program of the instrument can usually be used.
2. Data Analysis
Quantitative experiments should include at least three biological replicates. After the reaction, it is necessary to confirm the amplification curves and melting curves.
1) Amplification Curves:
The standard amplification curve should be S-shaped.
The most accurate quantitative analysis is achieved when the Ct value falls between 20 and 30.
If the Ct value is less than 10, the template should be diluted and the experiment should be repeated.
If the Ct value is between 30 and 35, the template concentration should be increased, or the reaction volume should be enlarged to improve amplification efficiency and ensure the accuracy of the results.
If the Ct value is greater than 35, the results cannot be used for quantitative analysis of gene expression but can be used for qualitative analysis.
2) Melting Curves:
A single peak in the melting curve indicates good specificity of the reaction and allows for quantitative analysis.
If the melting curve shows a double peak or multiple peaks, quantitative analysis cannot be performed.
If a double peak appears in the melting curve, DNA agarose gel electrophoresis should be used to determine whether the non-target peak is due to primer dimers or non-specific amplification.
If it is a primer dimer, it is recommended to reduce the primer concentration or redesign primers with higher amplification efficiency.
If it is non-specific amplification, the annealing temperature should be increased, or primers with higher specificity should be redesigned.
3. Primer Design Guidelines
1) The recommended primer length is around 25 bp. The optimal length of the amplicon is 150 bp, but it can be chosen within the range of 100 bp to 300 bp.
2) The Tm values of the forward and reverse primers should not differ by more than 2℃. A Tm value between 60℃ and 65℃ is preferred for primers.
3) The base distribution in the primer should be uniform, avoiding four consecutive identical bases. The GC content should be controlled around 50%. The last base at the 3’ end should preferably be G or C.
4) It is best to avoid complementary sequences of more than three bases within a primer or between the forward and reverse primers.
5) The specificity of the primers should be verified using the NCBI BLAST program. Avoid having more than two non-specific complementary bases at the 3’ end of the primers.
6) The designed primers need to be tested for amplification efficiency. Only primers with the same amplification efficiency should be used for quantitative comparative analysis.
4. Compatible Instruments
ABI: 5700, 7000, 7300, 7700, 7900HT Fast, StepOne, StepOne Plus; 7500, 7500 Fast, ViiA7, QuantStudio 3 and 5, QuantStudio 6,7,12k Flex;
Stratagene: MX3000P, MX3005P, MX4000P;
Bio-Rad: CFX96, CFX384, iCycler iQ, iQ5, MyiQ, MiniOpticon, Opticon, Opticon 2, Chromo4;
Eppendorf: Mastercycler ep realplex, realplex 2 s;
Qiagen: Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000;
Roche Applied Science: LightCycler 480, LightCycler 2.0; Lightcycler 96;
Thermo Scientific: PikoReal Cycler;
Cepheid: SmartCycler; Illumina: Eco qPCR.
