Multiplex One Step RT-qPCR Premix-UNG M2 HYA437
product detail
Multiplex One Step RT-qPCR Premix-UNG M2 is a dedicated reagent for qualitative and quantitative reactions via the one-step RT-PCR fluorescent probe method in multiplex amplification. The entire reaction is performed continuously in a single tube without tube opening, effectively preventing contamination.This product contains a thermostable reverse transcriptase with RNase H activity eliminated and a genetically engineered hot-start Taq DNA polymerase, delivering superior reverse transcription and PCR amplification efficiency. It is suitable for high-sensitivity amplification of low-concentration RNA templates.Formulated with an optimized qPCR-specific Buffer and the UNG/dUTP contamination prevention system, the reagent yields a high-quality standard curve over a broad quantification range for accurate quantification. It also effectively inhibits false-positive amplification caused by residual PCR products and aerosol contamination.This reagent is compatible with real-time fluorescent qPCR instruments from most manufacturers, including Applied Biosystems, Eppendorf, Bio-Rad and Roche.
Components
1. 20× RTase/UNG Multi Mix
2. 8×RT Premix Multi Buffer (dUTP) (Mg2+free)
3. 360mM MgCl2
Storage
Store at -20℃ for long-term storage. Stable for 3 months when stored at 4℃.Mix thoroughly before use. Avoid repeated freeze-thaw cycles.
Instructions
1. qRT-PCR Reaction System Preparation
|
Components |
25 uL Volume |
50 uL Volume |
Final Concentration |
|
8×RT Premix Multi Buffer (dUTP) (Mg2+ free) |
3.125 µL |
6.25 µL |
1× |
|
20× RTase/UNG Multi Mix |
1.25 µL |
2.5 µL |
1× |
|
360mM MgCl2 |
0.417 µL |
0.833 µL |
6 mM |
|
25×Primer-Probe Mix* |
1 µL |
2 µL |
1× |
|
Template RNA** |
—— |
—— |
—— |
|
ddH2O |
To 25 µL |
To 50 µL |
—— |
*A final primer concentration of 0.2 μM typically yields optimal results. If reaction performance is suboptimal, adjust the primer concentration within the range of 0.2–1 μM. The probe concentration is usually optimized between 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 RNA templates. Serial dilution may be performed if necessary to determine the optimal amount of RNA template to add.
2. Reaction program
|
Regular PCR program |
|||
|
Step |
Temperature |
Time |
Cycles |
|
Reverse transcription |
50℃ |
10-20 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: qRT-PCR sensitivity, specificity, and repeatability.
2. Free of extraneous nuclease activity, as well as extraneous endonuclease and exonuclease contamination.
Technical Information
1. This system contains TS-UNG enzyme, which can degrade uracil-containing DNA templates at room temperature (RT) or 25℃, and is inactivated during reverse transcription—thus not affecting the subsequent PCR amplification using cDNA as the template.
2. The reverse transcription reaction for this system is typically performed at 50℃. The reverse transcription temperature can be optimized within the range of 42–55℃; depending on the reaction characteristics, the reverse transcription time can be optimized between 5–30 minutes.
3. The RTase used in this system is genetically modified based on M-MLV, endowing it with higher temperature tolerance and a higher reverse transcription temperature. It exhibits superior reverse transcription efficiency for RNA templates with complex structures.
4. This system features excellent stability and applicability, making it highly suitable for the detection of complex templates such as viral RNA and RNA extracted from tissues. It delivers more stable amplification performance for extremely low-concentration templates, and is suitable for use in high-sensitivity molecular diagnostic reagents.
5. For primers with a low annealing temperature or the amplification of long fragments exceeding 200 bp, the three-step PCR protocol is recommended.
6. Different target genes vary in their dUTP utilization efficiency and sensitivity to UNG enzyme. Therefore, if the use of the UNG system results in a decrease in detection sensitivity, the reaction system should be adjusted and optimized.
7. Use dedicated areas and pipettes before and after amplification. Operate with gloves and replace them frequently. Do not open the reaction tubes after PCR completion to minimize contamination of samples by PCR products.
8. For Research Use Only!