Description
Product description
The 2×Color qPCR Fluore Green Master Mix (High Rox) is a 2× real-time quantitative PCR amplification premix, characterized by high fluorescence intensity, high sensitivity, strong specificity, and high amplification yield. The core component, Taq DNA polymerase, utilizes an antibody-mediated hot-start mechanism, which effectively inhibits non-specific amplification caused by primer annealing during sample preparation. Additionally, the mix contains factors that enhance PCR amplification efficiency and promote balanced amplification across a wide range of GC content (30-70%), ensuring good linearity in quantitative PCR over a broad quantification range.
This product employs a color-changing reaction based on the mixture of different dyes to monitor pipetting operations, effectively reducing the occurrence of pipetting errors.
Specifications
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Catalog Number
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N132036E
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N132036S
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Specifications
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50 T（20 μL/rxn）
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250 T（20 μL/rxn）
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Components
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Component Identification
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Component Name
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N132036E
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N132036S
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N132036-A
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2×Color qPCR Master Mix (High Rox)
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1 mL
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5×1 mL
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N132036-B
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10×Dilution Buffer
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1 mL
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1 mL
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Storage
Store at -25 to -15℃ in the dark. Valid for 1 year.
Notes
1. For your safety and health, please wear a lab coat and disposable gloves when operating.
2. After thawing, the Master Mix may appear with flocculent or white precipitates. Hold it in hand and gently dissolve by slowly inverting it up and down until the solution is clear. This does not affect the performance of the reagent.
3. Before use, invert the Master Mix gently to mix. Do not vortex to avoid generating bubbles, which may affect the quantification results. The Master Mix can be used after mixing and brief centrifugation. During pipetting, gently aspirate and dispense. If bubbles form in the Master Mix due to improper operation, centrifuge it again before use.
4. Due to the extremely high sensitivity of this product, it is susceptible to aerosol contamination in the air. Therefore, when preparing the reaction mixture, please do it in a laminar flow cabinet. Use sterile pipette tips and reaction tubes during preparation. Laboratories with the conditions are recommended to use dedicated pipettes and filter-tipped pipette tips.
5. It is recommended to use our company's cDNA synthesis kit (Cat#N132062) to effectively remove residual genomic material from RNA samples.
6. For Research Use Only.
Instructions
1. Template Processing
The 2×Color qPCR Fluore Green Master Mix (No Rox) contains a blue dye, and the 10×Dilution Buffer is a dedicated yellow concentrated template dilution solution. During use, if template tracing is required, dilute it to 1X as the template dilution solution and then proceed with qPCR detection. If template tracing is not required, simply do not use the Dilution Buffer.
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Template Types
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Usage of 10× Dilution Buffer
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Final Concentration
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cDNA solution, dissolved plasmid, genome
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If you need to dilute the template, first dilute the template to the desired concentration with sterile ultrapure water. Then, add 1 μL of 10×Dilution Buffer to every 9 μL of the diluted template.
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1×
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DNA Powder
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Dilute the 10×Dilution Buffer to 1× using sterile ultrapure water. Dissolve the corresponding DNA powder using an appropriate volume of 1× Dilution Buffer as the solvent.
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1×
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【Note】In actual use, other methods can also be employed as needed, as long as the final concentration of Dilution Buffer in the template is 1×.
2. Recommended qPCR Reaction System
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Component
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Volume（μL）***
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Volume（μL）***
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Final concentration
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2×miRNA qPCR Master Mix
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25
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10
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1×
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Forward Primer（self-provided）*
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1
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0.4
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0.2 μM
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Reverse Primer（10 μM）*
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1
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0.4
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0.2 μM
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cDNA **
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x
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x
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-
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RNase-free H2O
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Up to 50
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Up to 20
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-
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【Note】*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.
**If the template is undiluted cDNA stock solution, the volume used should not exceed 1/10 of the total qPCR reaction volume. The optimal amount of template added should result in Ct values between 20-30 cycles for amplification.
***A reaction volume of 20 μL or 50 μL is recommended to ensure the effectiveness and reproducibility of target gene amplification. Mix thoroughly before running the reaction to avoid excessive bubbles from vigorous shaking.
3. Reaction Program
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Recycling procedure
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Temperature
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Time
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Cycle Number
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Recycling procedure
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95℃
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2 min
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1
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Pre-denaturation
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95℃
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10 sec
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40
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Denaturation
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60℃
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30 sec**
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Melting Curve
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Default Settings
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1
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【Note】*Annealing Temperature and Time: Please adjust according to the primer Tm value and the length of the target gene.
**Fluorescence Signal Acquisition: Do not forget to enable fluorescence signal acquisition. Set up the experimental procedure according to the user manual of the instrument.
***Melting Curve: The default program of the instrument can usually be used.
4. Primer Design Guidelines
1) The recommended primer length is around 25 bp. The optimal amplicon size 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.
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) 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
5. Compatible Instruments
This product contains a pre-mixed ROX Reference Dye 1 (high concentration) for correcting well-to-well fluorescence signal errors, and is suitable for the following real-time fluorescent quantitative PCR instruments:
Applied Biosystems 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOnePlus;
And other real-time fluorescent quantitative PCR instruments that require the addition of high-concentration ROX Reference Dye.
