Superstart qPCR Premix plus-UNG HYA422
product detail
Superstart qPCR PreMix plus-UNG is specially developed for the lyophilization process. It provides components, e.g. Superstart Taq plus, uracil DNA glycosylase (UNG), MgCl2 , dNTPs (with dUTP instead of dTTP), and stabilizers, for one-step quantitative PCR (qPCR). Superstart Taq plus, novelly modified, provides an automatic “hot start”in PCR for increased sensitivity, specificity, and yield, and has a short activation time. Good calibration curve can be achieved under wide quantitative range due to the optimized qPCR buffer, and thus inhibiting false positive amplification caused by PCR residual and aerosol pollution. This reagent is compatible with most fluorescent quantitative PCR instruments, such as Applied Biosystems, Eppendorf, Bio-Rad, Roche and so on. After lyophilization, the reagent maintains good stability.
Components
1. 5×Superstart Premix plus-UNG (Mg 2+free) (DG)
2. 250mM MgCl2
3. 4×Lyoprotectant (Optional)
Storage
Long-term storage at -20℃ ; can be stored at 4℃ for up to 3 months. Mix well before use and avoid repeated freezing and thawing.
Instructions
1. Reaction system
|
Components |
25µL Volume |
50µL Volume |
Final Concentration |
|
5×Superstart Premix plus-UNG (Mg 2+ free) (DG) |
5 µL |
10 µL |
1× |
|
250mM MgCl2 |
0.45 µL |
0.9 µL |
4.5 mM |
|
4×lyoprotectant* |
6.25 µL |
12.5 µL |
1× |
|
25×Primer-Probe Mix** |
1 µL |
2 µL |
1× |
|
Template DNA*** |
- |
- |
- |
|
ddH2O |
Up to 25 µL |
Up to 50 µL |
- |
1. This system can be lyophilized; when there is no need to do so, 4×Lyoprotectant can be selectively added during use. If there is a need to produce lyophilized products, during liquid testing stage verification of product performance must include 4 × Lyoprotectant to ensure consistency with the composition and effects after
2. Typically 0.2 μM primer concentration yields better results; if reaction performance is poor then adjust primerconcentration between 0.2~1 μ M range accordingly. Typically probe concentrations are optimized within the range of 0.1~0.3 μ M. Concentration gradient experiments may be performed to find the best combination of primers and probes.
3. The copy number of target genes contained in different types of templates differs; therefore, it may benecessary to perform gradient dilution experiments to determine the optimal amount of template addition.
2. Reaction program
|
Step |
Temperature |
Time |
Cycles |
|
Digestion |
50℃ |
2 min |
1 |
|
Polymerase activation |
95℃ |
1-5 min |
1 |
|
Denaturation |
95 ℃ |
10-20 s |
40-50 |
|
Annealing and Extension |
56~64℃ |
20-60 s |
*Annealing/extension temperature can be adjusted appropriately according to experimental requirements.
Lyophilization system preparation
|
Component |
Volume |
|
5×Superstart Premix plus-UNG (Mg 2+ free) (DG) |
5 µL |
|
250mM MgCl2 |
1 µL |
|
4×lyoprotectant* |
6.25 µL |
|
25×Primer-Probe Mix** |
1 µL |
|
ddH2O |
Up to 18-20 µL |
*The concentration of primer probe can be adjusted appropriately according to the experimental requirements, and the total volume cannot exceed 5 μL.
**Primer probe can be added before freeze drying, depending on the experimental plan; if primer probe is not added during freeze drying in this step, it must be added in "3. Reaction amplification system".
Lyophilization Process
|
Step |
Temperature |
Set time |
Condition |
Pressure |
|
Freezing |
4℃ |
30 min |
Hold |
1 atm |
|
-50℃ |
60 min |
Ramp |
||
|
-50℃ |
180 min |
Hold |
||
|
Primary Drying |
-30℃ |
60 min |
Ramp |
Ultimate Vacuum |
|
-30℃ |
720 min |
Hold |
||
|
Secondary Drying |
25℃ |
60 min |
Ramp |
Ultimate Vacuum |
|
25℃ |
300 min |
Hold |
This cycle protocol is suitable for in-situ lyophilization. If you need to produce lyophilized microspheres, please contact us for technical support. The parameters provided are for reference only and should be optimized to different lyophilized product formats and machines.
Guidelines for lyophilized products (in PCR tube)
1. Centrifuge the lyophilized powder instantaneously.
2. Add the template to the lyophilized powder, and add water up to 25 μL/50 μL.
3. Mix and centrifuge, and then proceed with PCR amplification
* It is recommended to use Biori integrated mixer-centrifuge CM-8 or CM-8 Plus for standardized mixing and centrifugation operations.
Quality Control
1. Function detection: sensitivity, specificity and repeatability of qPCR.
2. No exogenous nuclease activity: no exogenous endonuclease and exonuclease pollution.
Technical Information
1. This product employs a novel type of hot-start DNA polymerase, which can be activated in 1-5 minutes. Since itsreaction buffer has been specially optimized, it is more suitable for double or multiple fluorescence quantitative PCR containing internal standard genes.
2. This reagent performs high specificity, and significantly improves the sensitivity of fluorescence quantitativePCR, as well as the normalization of the amplification curve and fluorescence value of extremely low concentration templates. The kit is recommended for high-sensitivity fluorescence quantitative PCR detection.
3. Three-step PCR method is recommended for primers with low annealing temperature or for amplification oflong fragments over 200 bp.
4. Since different amplicons have different utilization efficiency to dUTP and different sensitivity to UNG, thereagents should be optimized if the detection sensitivity decreases when using UNG system. Please contact us for technical support if needed.
5. To avoid amplification of carryover PCR products, dedicated experimental area and pipette are required for Operate with gloves and change frequently and do not open the PCR tube after amplification.