Description
Hifair™ V Multiplex One Step RT-qPCR Probe Kit (UDG Plus) is a multiplex quantitative PCR kit based on RNA as a 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 Hifair™ V Reverse Transcriptase and quantitatively amplified by Unicon™ 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 reactions. The dUTP/UDG system was added to effectively prevent the risk of aerosol contamination.
Features
- Hot start chemical reagents provide room temperature stability.
- Complete RT and qPCR steps in a single tube - for convenience and reduced risk of contamination.
- qPCR was performed using the entire RT reaction as a template -improved sensitivity.
Applications
- Detection of pathogens
- Diagnosis and research of tumor
- Animal disease detection
- Early diagnosis of hereditary diseases
- Detection of pathogenic microorganisms in food
Specifications
| Hot Start | Built-in hot start |
| Detection method | Primer-probe detection |
| PCR method | One step RT-qPCR |
| Type of sample | RNA |
| Application equipment | Equipment with Rox: ABI 5700, 7000, 7300, 7700, 7900HT Fast, StepOne™, StepOne Plus™ Equipment with Low Rox: ABI 7500, 7500 Fast, ViiA™7, QuantStudio™ 3 and 5, QuantStudio™ 6,7,12k Flex Stratagene MX3000P™, MX3005P™, MX4000P™ Equipment without Rox: Bio-Rad CFX96™, CFX384™, iCycler iQ™, iQ™5, 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 |
Components
|
Components No. |
Name |
13650ES50 (50 T) |
13650ES60 (100 T) |
13650ES80 (1,000 T) |
|
13650-A |
2×Hifair™ V MP Buffer |
750 μL |
1.5 mL |
15 mL |
|
13650-B |
Hifair™ V Enzyme Mix |
60 μL |
120 μL |
1.2 mL |
Note: 1) 2×Hifair™ V MP Buffer is the abbreviation of Hifair™ V Multiplex One Step RT-qPCR Probe Buffer, which includes dNTPs, dUTP, Mg2+, stabilizers, enhancers, and more.
2) Hifair™ V Enzyme Mix mainly contains heat-resistant Hifair™ V reverse transcriptase, UNICON™ HotStart Taq DNA polymeras and UDGase.
Instructions
- Reaction Composition
|
Components |
Volume (μL) |
Final Concentration |
|
2×Hifair™ V MP Buffer |
15 |
1× |
|
Hifair™ V Enzyme Mix |
1.2 |
- |
|
Primer / Probe Mix (2.5 μM) |
3 |
0.25 μM |
|
Template RNA |
1-10 |
- |
|
RNase Free H2O |
to 30 |
- |
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.
Cycle Setup
Regular
| CYCLE STEP |
TEMPERATURE |
TIME | CYCLES |
| Reverse Transcription |
50°C* | 20 mins |
1 |
| Initial Denaturation |
95°C | 5 mins |
1 |
| Denaturation Extension |
95°C 60°C |
15 secs 30 secs |
40-45 |
Fast 1
| CYCLE STEP |
TEMPERATURE |
TIME | CYCLES |
| Reverse Transcription |
50°C* | 10 mins |
1 |
| Initial Denaturation |
95°C | 1 min |
1 |
| Denaturation Extension |
95°C 60°C |
10 secs 30 secs |
40-45 |
Fast 2**
| CYCLE STEP |
TEMPERATURE |
TIME | CYCLES |
| Reverse Transcription |
50°C* | 2 mins |
1 |
| Initial Denaturation |
95°C | 2 secs |
1 |
| Denaturation Extension |
95°C 60°C |
1 sec 8 secs |
40-45 |
*50°C is optimal for the Hifair™ Reverse Transcriptase. To ensure best performance, avoid using a temperature of < 50°C and ＞55°C .
** “Fast” ramp speed mode can be used where available (e.g., QuantStudio5 and so on).Shipping and Storage
The product is shipped with dry ice and can be stored at -15℃ ~ -25℃ for 1 year. It should avoid repeated freezing and thawing. It is recommended to save separately.
Figures
Figure 1 High stability: 37℃ thermal acceleration stability.
Figure 2 High stability: freeze-thaw stability.
