Multiplex One Step RT-qPCR Premix
Description
Cat No: HCR5141A
Multiplex One Step RT-qPCR Premix is a multiplex quantitative PCR Kit based on RNA as template. In the experiment, reverse transcription and quantitative PCR are performed in the same reaction tube, simplifying the experimental operation and reducing the risk of contamination. The unique design of buffer and enzyme mix can be used in one-step lyophilized system. The kit utilizes heat-resistant Reverse Transcriptase for efficient synthesis of first strand cDNA using hotstart Taq DNA Polymerase for quantitative amplification. It contains optimized reaction buffer, enzymes mix etc., and factors that effectively inhibit non-specific PCR amplification and enhance the amplification efficiency of multiple qPCR reactions were added, enabling multiple fluorescence quantitative amplification while ensuring the amplification efficiency of primers.
Components
|
Name |
|
1. Lyo-Buffer |
|
2. Lyo-Enzyme Mix |
|
3. Lyo protectant |
Transportation condition
A: Lyo-buffer and protectant: -25~-15℃, shelf life is 1 year.
B: Lyo-enzyme mix, 2-8 ℃, shelf life is 6 months.
Instruction for operation
1. Reaction System (Take 25μL as example )
|
Components |
Volume (μL) |
Final Concentration |
|
Lyo-Buffer |
6 |
1* |
|
Lyo-Enzyme Mix |
1 |
- |
|
Lyo-Protectant |
8 |
- |
|
Primer Mix (10μM) |
1 |
0.1- 1uM |
|
Probe Mix (10μM) |
0.5 |
0.05-0.5uM |
|
RNA Template |
5 |
- |
|
DEPC H2O |
Up to 25 |
- |
2. Optimized Cycling Protocol
1) Standard Cycling Protocol
|
Reaction stage |
Temperature |
Time |
Cycle |
|
|
1 |
Reverse transcription |
50°C a |
10min |
1 |
|
2 |
Initial denaturation |
95°C |
5min |
1 |
|
3 |
Amplification reaction |
95°C |
15sec |
45 cycles |
|
60°C b |
30sec c |
2) Fast Cycling Protocol
|
|
Reaction stage |
Temperature |
Time |
Cycle |
|
1 |
Reverse transcription |
50°C a |
2min |
1 |
|
2 |
Initial denaturation |
95°C |
2sec |
1 |
|
3 |
Amplification reaction |
95°C |
1sec |
45 cycles |
|
60°C b |
13sec c |
Note:
a) Reverse transcription: The temperature can select 42°C or 50°C for 10-15 minutes.
b) Amplification reaction: The temperature is adjusted according to the Tm value of the designed primers.
c) Fluorescence signal acquisition: Please set the experimental procedure according to the requirements of the instrument manual.
Technical Information/Specifications
|
Hot Start |
Built-in hot start |
|
Detection method |
Primer-probe detection |
|
PCR method |
One step RT-qPCR |
|
Type of sample |
RNA |
Notes
1. This product is for research use only.
2. Please wear the necessary PPE, such lab coat and gloves, to ensure your health and safety！
