Description
Product description
The 2×miRNA qPCR Fluore Green Master Mix is a new-generation premixed fluorescent quantitative PCR detection reagent specifically developed for miRNA quantification. It contains a special ROX Passive Reference Dye, which is compatible with all qPCR instruments without the need to adjust the ROX concentration for different instruments. The DNA Polymerase used is a chemically modified hot-start polymerase, combined with a special buffer system, which enhances the specificity of the reaction, improves sensitivity, and allows for accurate quantification over a broader range.
This product must be used in conjunction with our miRNA First-Strand cDNA Synthesis Reverse Transcription Kit (Poly(A) tailing method, for qPCR) (Cat#N132068) to obtain the final experimental results.
Specifications
|
Catalog Number
|
N132039E
|
N132039S
|
|
Specifications
|
100 T（20 μL/rxn）
|
500T（20 μL/rxn）
|
Components
|
Component Identification
|
Component Name
|
N132036E
|
N132036S
|
|
N132039-A
|
2×miRNA qPCR Master Mix
|
1 mL
|
5×1 mL
|
|
N132039-B
|
RNase-free H2O
|
2×1 mL
|
5×1 mL
|
Storage
Ice bag transportation. Store at -20℃ in the dark. Valid for 12 months.
Notes
1. Thaw the components completely and gently mix them before use.
2. During the experiment, use contamination-free consumables whenever possible to avoid contamination.
3. Avoid repeated freeze-thaw cycles for this product. Protect from strong light during preparation.
4. For your safety and health, please wear a lab coat and disposable gloves when operating.
5. This product is for research use only.
Instructions
1. Reaction Preparation
1) Place the reagents at room temperature to thaw. Before use, invert the reagents gently to mix and then briefly centrifuge to collect any residual liquid from the cap. Avoid vigorous shaking or vortexing to prevent foaming.
2) Keep the reagents on ice and prepare the reaction mixture according to the table below. RNase-free H2O can be substituted with other nuclease-free water used for molecular biology experiments.
【Note】Failure to mix the reagents properly, using a vortex mixer, or not preparing the reagents on ice can lead to decreased reaction performance.
Preparation of Quantitative PCR Reaction Mixture
|
Component
|
Volume（μL）
|
Volume（μL）
|
Final concentration
|
|
2×miRNA qPCR Master Mix
|
10 μL
|
25 μL
|
1 ×
|
|
Forward Primer（self-provided）
|
X
|
X
|
400 nM
|
|
Reverse Primer (10 μM)
|
0.8 μL
|
2 μL
|
400 nM
|
|
cDNA
|
X
|
X
|
-
|
|
RNase-free H2O
|
Up to 20 μL
|
Up to 50 μL
|
-
|
【Note】a. The volume of miRNA first-strand cDNA added should not exceed 1/10 of the total qRT-PCR reaction volume. High concentrations of cDNA may lead to non-specific amplification; it is recommended to dilute the cDNA 10-1000 times as appropriate. b. The Reverse Primer (10 μM) is sourced from Kit N132068. Due to patent confidentiality, the downstream primer sequence for miRNA poly(A) tailing reverse transcription may vary between manufacturers. It is recommended to use Kit N132068 to ensure reliable experimental results.
2. PCR Reaction Program Setup
You can refer to the following two programs for quantitative PCR reactions.
1) Conventional Fluorescent Quantitative PCR Amplification Program (Two-Step Method)
|
Recycling procedure
|
Temperature
|
Time
|
Cycle Number
|
|
Pre-denaturation
|
95℃
|
10 min
|
1
|
|
Denaturation
|
95℃
|
15 sec
|
35-40
|
|
Annealing/extension
|
60℃
|
20 sec
|
|
Melting Curve Analysis
|
Default Settings
|
1
|
2) Fluorescent Quantitative PCR Fast Amplification Program (Two-Step Method)
|
Recycling procedure
|
Temperature
|
Time
|
Cycle Number
|
|
Pre-denaturation
|
95℃
|
10 sec
|
1
|
|
Denaturation
|
95℃
|
5 sec
|
40
|
|
Annealing/extension
|
60℃
|
20 sec
|
|
Melting Curve Analysis
|
Default Settings
|
1
|
【Note】The annealing/extension temperature and time can be adjusted according to experimental requirements.The choice between the conventional and fast programs depends on the PCR instrument being used. For example, instruments like the ABI QuantStudio 5 can be set to a fast program, while instruments like the Bio-Rad CFX96 do not have a fast program setting and require the conventional program to be used.
