RobuDirect Probe RT-qPCR Premix plus-UNG HYA438
product detail
The 2x One-Step RT-qPCR Premix-UNG is a dedicated reagent for qualitative and quantitative reactions using one-step RT-PCR fluorescence quantitative probe method. This product utilizes directionally modified high-affinity antibody-modified DNA amplification enzymes and warm-start reverse transcriptases. Through deep optimization of the buffer, it achieves complete premixing of enzymes, buffer, and primers/probes, ensuring stability during long-term low-temperature storage and improving the ease of use of the final product. The reagent incorporates a dUTP/UNG anti-contamination system, effectively preventing false positive reactions caused by PCR product residue and aerosol contamination, ensuring accurate results.
Components
1. 25×RTase/UNG Mix
2. 2×RT Premix Buffer (dUTP)
Storage
Store at -20℃ for extended periods; at 4℃ for up to 3 months. Mix well before use and avoid repeated freeze-thaw cycles.
Instructions
1. qRT-PCR Reaction System Preparation
|
Components |
25 uL Volume |
50 uL Volume |
Final Concentration |
|
2× RT Premix Buffer (dUTP) |
12.5 µL |
25 µL |
1× |
|
25× RTase/UNG Mix |
1 µL |
2 µL |
1× |
|
25×Primer-Probe Mix* |
1 µL |
2 µL |
1× |
|
Template RNA** |
—— |
—— |
—— |
|
ddH2O |
To 25 µL |
To 50 µL |
—— |
*Generally, a final primer concentration of 0.2 μM yields good results; for poor reaction performance, the primer concentration can be adjusted within the range of 0.2–1 μM. The probe concentration is typically optimized within the range of 0.1–0.3 μM. Concentration gradient experiments can be performed to find the optimal combination of primers and probes.
**Different types of RNA templates contain different copy numbers of the target gene; if necessary, serial dilutions can be performed to determine the optimal amount of RNA template added.
2. Reaction program
|
Step |
Temperature |
Time |
Cycles |
|
Reverse transcription |
50℃ |
5-10 min |
1 |
|
Pre-denaturation |
95℃ |
30 s-1 min |
1 |
|
Denaturation |
95 ℃ |
10-20 s |
40-50 |
|
Annealing and Extension |
56~64℃ |
20-60 s |
* The temperature-sensitive TS-UNG enzyme contained in this product can perform digestion at room temperature and become inactive during the reverse transcription process.
Quality Control
1. Function detection: sensitivity, specificity and repeatability of qRT-PCR.
2. No exogenous nuclease activity: no exogenous endonuclease and exonuclease pollution.
Technical Information
1. This system contains TS-UNG enzyme, which degrades uracil-containing DNA templates at room temperature or 25°C and is inactivated during reverse transcription, thus not affecting subsequent PCR amplification using cDNA as a template.
2. This system commonly uses 50°C for reverse transcription, but the temperature can be optimized within the range of 42°C to 55°C. Depending on the reaction characteristics, the reverse transcription time can be optimized within 5 to 30 minutes.
3. The novel reverse transcriptase used in this system is a genetically modified version of M-MLV, giving it higher temperature tolerance and reverse transcription temperature, resulting in higher reverse transcription efficiency for complex RNA templates.
4. This system exhibits good stability and applicability, making it ideal for detecting viruses and complex RNA templates extracted from tissues. It provides more stable amplification results even with extremely low template concentrations, making it suitable for use with high-sensitivity molecular diagnostic reagents.
5. For primers with lower annealing temperatures or amplification of fragments longer than 200 bp, a three-step method is recommended.
6. Different genes to be amplified have varying utilization efficiency of dUTP and sensitivity to UNG enzymes. Therefore, if using the UNG system leads to a decrease in detection sensitivity, the reaction system should be adjusted and optimized. Please contact our company for technical support.
7. Please use dedicated regions and pipettes before and after amplification, wear gloves during operation, and change gloves frequently. Do not open the reaction tube after the PCR reaction is complete to minimize PCR product contamination of the sample.
8. To avoid amplification of carryover PCR products, dedicated experimental area and pipette are required for amplification. Operate with gloves and change frequently and do not open the PCR tube after amplification.