Description
Hifair Multiplex One Step RT-qPCR Probe Kit (UDG Plus) is a multiplex quantitative PCR kit based on RNA as template. In the process of the experiment, reverse transcription and quantitative PCR were carried out in the same tube, which simplified the experimental operation and reduced the risk of contamination.
In this kit, the first strand cDNA was efficiently synthesized by heat-resistant HifairTM V Reverse Transcriptase and quantitatively amplified by UNICONTM HotStart Taq DNA Polymerase. The kit mainly contains optimized MP buffer, enzymes mix, etc. The buffer solution already contains Mg2+ and dNTP. In addition, the factors that can effectively inhibit the non-specific PCR amplification and improve the amplification efficiency of multiple qPCR reactions are added, which can ensure the amplification efficiency and carry out up to multiple amplification reaction. The dUTP/UDG system was added to effectively prevent the risk of aerosol contamination.
Components
|
Components |
Name |
16630ES60 (100 T) |
16630ES80 (1000 T) |
16630ES92 (10000 T) |
|
16630-A |
5×Hifair MP Buffer |
500 μL |
5 mL |
50 mL |
|
16630-B |
Hifair Enzyme Mix |
100 μL |
1 mL |
10 mL |
|
16630-C |
GC-Enhancer |
500 μL |
5 mL |
50 mL |
Note: 1) 5×Hifair MP Buffer is the abbreviation of HifairTM V Multiplex One Step RT-qPCR Probe Buffer, which includes dNTPs, dUTP, Mg2+, stabilizers, enhancers, and more.
2) Hifair Enzyme Mix mainly contains heat-resistant HifairTM V reverse transcriptase, UNICONTM HotStart Taq DNA polymeras and UDGase.
3) GC enhancer is optional
Specifications
Figure
Universal Application
Sensitivity
Stabililty
Storage
The product should be stored at -25°C~-15°C for 1 year.
Instructions
1. Reaction Composition
|
Components |
Volume (μL) |
Final Concentration |
|
5×Hifair MP Buffer |
5 |
1 × |
|
Hifair Enzyme Mix |
1 |
- |
|
GC-Enhancer |
0-5 |
- |
|
Primer Mix (10 μM) |
0.4 each |
0.16 μM |
|
Probe Mix (10 μM) |
0.2 each |
0.08 μM |
|
RNA |
1-5 |
- |
|
RNase Free H2O |
to 25 |
- |
Note：Be sure to mix well before use, avoid excessive bubbles caused by violent vibration.
a) Primer concentration: Primer mix including multiplex primer, depending on the situation optimal primer concentration may be between 0.1 and 1.0 μM.
b) Probe concentration: Probe mix including multiplex probe labeling difference fluorescent group, depending on the situation optimal probe concentration may be between 0.05 and 0.5 μM.
c) Template dilution: qPCR is highly sensitive and it is recommended to dilute the template. The control Ct value is suitable between 20 and 35.
d) System preparation: un head with filter element. Avoid cross contamination and aerosol contamination.
2. Optimized Cycling Protocol
|
Reaction stage |
Temperature |
Time |
Cycle |
|
|
1 |
Reverse transcription |
50°Ca |
15 min |
1 |
|
2 |
Initial denaturation |
95°C |
5 min |
1 |
|
3 |
Amplification reaction |
95°C |
15 sec |
45 cycles |
|
60°Cb |
30 secc |
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.