FastAmpli Probe qPCR Premix plus-UNG HYA423
product detail
FastAmpli Probe qPCR Premix plus-UNG is a specialized reagent for qualitative and quantitative Real Time PCR (RT-qPCR) reactions using the probe-based method. It contains a genetically engineered and selected rapid-amplification DNA polymerase, enabling PCR reactions to be completed within 30 minutes. With matched optimization of the Buffer system, this product is suitable for multiplex amplification. Formulated with a mixture of inhibition-resistant amplification enzyme and UNG enzyme, along with an optimized Buffer system containing dUTP, this reagent not only achieves efficient amplification of target genes in samples containing inhibitors but also effectively prevents false-positive reactions caused by PCR product carryover and aerosol contamination. It is compatible with real-time PCR instruments from most manufacturers, including Applied Biosystems, Eppendorf, Bio-Rad, and Roche.
Components
2×FastAmpli Probe qPCR Premix plus-UNG
*This reagent already contains components such as hot-start DNA polymerase, UNG, PCR buffer, MgCl2, dNTPs, and stabilizers.
Storage
Store at -20℃ for long-term storage; stable for 3 months at 4℃. Mix thoroughly before use and avoid repeated freeze-thaw cycles.
Instructions
1. Reaction system
|
Components |
25µL Volume |
50µL Volume |
Final Concentration |
|
2×FastAmpli Probe qPCR Premix plus-UNG |
12.5 µL |
25 µL |
1× |
|
25×Primer-Probe Mix* |
1 µL |
2 µL |
1× |
|
Template DNA** |
- |
- |
- |
|
ddH2O |
Up to 25 µL |
Up to 50 µL |
- |
*When using conventional PCR protocols for amplification, a final primer concentration of 0.2 μM typically yields satisfactory results; if the reaction performance is unsatisfactory, the primer concentration can be adjusted within the range of 0.2–1 μM. Generally, the probe concentration is optimized within the range of 0.1–0.3 μM. Concentration gradient experiments can be performed to identify the optimal combination of primers and probes.When using rapid PCR protocols for amplification, appropriately increasing the concentrations of primers and probes may yield better amplification results, and the primer-probe ratio should undergo ratio optimization.
**The copy number of the target gene varies among different types of templates. If necessary, serial dilution can be conducted to determine the optimal amount of template to add.
2. Reaction program
|
Regular PCR program |
|||
|
Step |
Temperature |
Time |
Cycles |
|
Digestion |
50℃ |
2 min |
1 |
|
Pre-denaturation |
95℃ |
1-5 min |
1 |
|
Denaturation |
95 ℃ |
10-20 s |
40-50 |
|
Annealing and Extension |
56~64℃ |
20-60 s |
|
|
Rapid PCR program |
|||
|
Step |
Temperature |
Time |
Cycles |
|
Digestion |
50℃ |
2 min |
1 |
|
Pre-denaturation |
95℃ |
30 s |
1 |
|
Denaturation |
95 ℃ |
1-3 s |
40-45 |
|
Annealing and Extension |
56~64℃ |
3-20 s |
|
Quality Control
- Functional Testing: qPCR sensitivity, specificity, and repeatability.
- Free of extraneous nuclease activity, as well as extraneous endonuclease and exonuclease contamination.
Technical Information
1. The amplification rate of the rapid DNA polymerase contained in this product is no less than 1 kb/10 s. Significant variations exist in temperature ramping rate, temperature control mode, and heat transfer efficiency among different rapid PCR instruments. It is recommended to optimize the optimal reaction parameters in conjunction with the specific rapid PCR instrument used.
2. It exhibits enhanced specificity and significantly improves the sensitivity for limit detection in real-time PCR. This results in marked improvements in the normalization of amplification curves and fluorescence values for ultra-low concentration templates, making it suitable for use as a high-sensitivity real-time PCR detection reagent.
3. For primers with low annealing temperatures or amplification of long fragments exceeding 200 bp, the three-step PCR protocol is recommended.
4. Different target genes vary in their dUTP utilization efficiency and sensitivity to UNG enzyme. Therefore, if the use of the UNG system leads to decreased detection sensitivity, the reaction system should be adjusted and optimized accordingly.
5. Use dedicated areas and pipettes for pre- and post-amplification steps. Operate with gloves and replace them frequently. Do not open the reaction tubes after PCR completion to minimize contamination of samples by PCR amplicons.
6. For Research Use Only !