Superstart Probe qPCR Premix plus-UNG HYA422
product detail
Superstart Probe qPCR Premix plus-UNG is a specialized reagent designed for qualitative and quantitative Real Time PCR (RT-qPCR) reactions using the probe-based method. It contains Superstart Taq plus, a novel hot-start enzyme with blocked Taq polymerase activity at room temperature, which effectively inhibits nonspecific amplification caused by primer nonspecific annealing or primer dimers under low-temperature conditions. This enhances the specificity of amplification reactions and delivers more stable detection results for ultra-low concentration templates. Formulated with an optimized qPCR-specific Buffer and a UNG/dUTP anti-contamination system, this reagent generates excellent standard curves over a wide quantitation range, enabling accurate quantification. It effectively prevents false-positive amplification induced by PCR product carryover and aerosol contamination. This reagent is compatible with real-time PCR instruments from most manufacturers, including Applied Biosystems, Eppendorf, Bio-Rad, and Roche.
Components
2×Superstart Probe qPCR Premix plus-UNG
*This reagent already contains Superstart Taq plus, UNG, PCR Buffer, MgCl2, dNTPs, stabilizers, and other components.
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×Superstart 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 |
- |
*Typically, a final primer concentration of 0.2 μM 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.
**The copy number of the target gene varies among different types of DNA templates. If necessary, serial dilution can be conducted to determine the optimal amount of DNA template to add.
2. Reaction 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 |
Quality Control
1. Functional Testing: qPCR sensitivity, specificity, and repeatability.
2. Free of extraneous nuclease activity, as well as extraneous endonuclease and exonuclease contamination.
Technical Information
1. Superstart Probe qPCR Premix plus-UNG incorporates a novel hot-start enzyme, enabling rapid hot start within 1–5 minutes. Formulated with a specially optimized Buffer system, it is compatible with multiplex real-time PCR reactions.
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 !