| Product Description | UltraSYBR Mixture is a premixed system specifically designed for real-time fluorescence quantitative PCR using the dye method (SYBR Green I), with a concentration of 2 x, containing GoldStar Taq DNA Polymerase, PCR Buffer, dNTPs, SYBR Green I fluorescent dye, and Mg2+. The operation is simple and convenient. Mainly used for detecting genomic DNA target sequences and cDNA target sequences after RNA reverse transcription.
The fluorescent dye SYBR Green I contained in this product can bind to all double stranded DNA, making it suitable for detecting different target sequences without the need for synthesizing specific labeled probes. Among them, GoldStar Taq DNA Polymerase is a chemically modified, novel and highly efficient hot start enzyme that has no polymerase activity at room temperature, effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature. Enzyme activation requires incubation at 95 ℃ for 10 minutes. The unique combination of PCR buffer system and hot start enzyme effectively inhibits non-specific PCR amplification and significantly improves the amplification efficiency of PCR. This product is suitable for fluorescence quantitative PCR instruments that do not require ROX as a correction dye, such as Roche LightCyster 480, Roche LightCyler 96, Bio rad iCyler iQ, iQ5, CFX96. | U665891 | Component | 5 mL | 40 mL | Storage | | U665891A | 2×Ultra SYBR Mixture | 5×1 mL | 40×1 mL | -20°C. Avoid freeze/thaw cycle.
| | U665891B | ddH₂O | 5×1 mL | 40×1 mL | -20°C. Avoid freeze/thaw cycle |
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Product features
1. This product uses a new and efficient hot start enzyme GoldStar Taq DNA Polymerase and a unique PCR buffer system, significantly improving the amplification efficiency of PCR with high sensitivity and strong specificity.
2. Suitable for fluorescence quantitative PCR detection, it can accurately quantify and detect target genes.
Matters needing attention
1. Before use, please gently mix upside down to avoid foaming, and use after briefly centrifugation.
2. This product contains SYBR Green I fluorescent dye. When storing this product or preparing PCR reaction solution, avoid strong light exposure.
3. Avoid repeated freezing and thawing of this product, as repeated freezing and thawing may cause a decrease in product performance.
4. This product cannot be used for probe based fluorescence quantitative PCR.
When preparing the reaction solution, please use new or non polluting gun heads and centrifuge tubes to prevent contamination as much as possible. Usage
The following are examples of conventional PCR reaction systems and reaction conditions. In practical operation, corresponding improvements and optimizations should be made based on different templates, primer structures, and target fragment sizes.
1. PCR reaction system
| Reagent |
50 μL Reaction system |
Final concentration |
| 2×UltraSYBR Mixture |
25 μL |
1 × |
| Forward Primer，10 µM |
1 μL |
0.2 μM ¹⁾ |
| Reverse Primer，10 µM |
1 μL |
0.2 μM ¹⁾ |
| Template DNA |
2 μl ²⁾ |
/ |
| ddH2O |
up to 50 μl |
/ |
|
Attention:
1) Typically, the primer concentration is 0.2 μ M can obtain good results, with a final concentration of 0.1-1.0 μ M serves as a reference for setting the range.
2) The amount of DNA template is usually based on 10-100 ng genomic DNA or 1-10 ng cDNA as a reference. Due to the different copy numbers of target genes contained in templates of different species, gradient dilution can be applied to the template to determine the optimal template usage.
3) The recommended reaction system is 50 μ l. The reaction system can also be scaled up or down proportionally according to actual experimental needs.
2. PCR reaction program:
Attention! The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes!
It is recommended to use the two-step PCR method shown in the table below for program setting. This program takes the ABI7500 fluorescence quantitative PCR instrument as an example. If good experimental results cannot be obtained due to primers with low Tm values, a three-step PCR amplification method can be attempted. The operation steps of the three-step method are detailed in "Optimization of Reaction Conditions".
| Step |
Temperature |
Time |
/ |
| Pre denaturation |
95℃ |
10 min ¹⁾ |
/ |
| Denaturation |
95℃ |
15 s |
35-40 cycles |
| Annealing/Extension ² ⁾ |
60℃ |
1 min |
35-40 cycles |
| Analysis of Fusion Curve ³ ⁾ |
/ |
/ |
/ |
| / |
95℃ |
15 s |
/ |
| / |
60℃ |
1 min |
/ |
| / |
95℃ |
15 s |
/ |
| / |
60℃ |
15 s |
/ |
|
Attention:
1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.
2) The annealing temperature should be set at 60-64 ℃ as a reference range. When non-specific reactions occur, the annealing temperature can be increased.
3) This program is based on the ABI 7500 fluorescence quantitative PCR instrument as a reference setting. For melting curve analysis, please use the recommended program for the fluorescence quantitative PCR instrument used.
Optimization of reaction conditions
When optimizing fluorescence quantitative reaction conditions, factors such as primer concentration, annealing temperature, and extension time should be considered to improve reaction specificity and amplification efficiency.
1. An experimental system with high reaction specificity and amplification efficiency should meet the following conditions:
1) High reaction specificity: negative control without non-specific amplification such as primer dimer; No amplification beyond the target fragment is generated.
2) High amplification efficiency: low Ct value; The PCR amplification efficiency is high, approaching the theoretical value of 100%.
2. Optimization method for reaction conditions:
1) Primer concentration: Typically, the primer concentration is 0.2 μ M can obtain good results, with a final concentration of 0.1-1.0 μ M serves as a reference for setting the range. If the reaction specificity is increased, the primer concentration can be reduced; If the amplification efficiency is improved, the concentration of primers can be increased, thereby optimizing the reaction system.
2) Annealing temperature: It is recommended to use two-step PCR with an annealing temperature of 60 ℃ for reaction. If the reaction specificity is improved, the annealing temperature can be increased, with 60-64 ℃ as the reference range for setting. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification method can be attempted. The annealing temperature of the three-step method should be set within the range of 56 ℃ -64 ℃ as a reference.
3) Extension time: It is recommended to use two-step PCR with an extension time of 1 minute for the reaction. If the amplification efficiency is improved, it can be attempted to increase the extension time or try the three-step PCR method Attention! The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes! Three step fluorescence quantitative PCR (this program takes the ABI7500 fluorescence quantitative PCR instrument as an example):
| Step |
Temperature |
Time |
/ |
| Pre denaturation |
95℃ |
10 min ¹⁾ |
/ |
| Denaturation |
95℃ |
10 s |
35-40 cycles |
| Annealing |
56-64℃ ²⁾ |
30 s |
35-40 cycles |
| Extension |
72℃ |
32 s ³ ⁾ |
35-40 cycles |
| Analysis of Fusion Curve ⁴⁾ |
/ |
/ |
/ |
| / |
95℃ |
15 s |
/ |
| / |
60℃ |
1 min |
/ |
| / |
95℃ |
15 s |
/ |
| / |
60℃ |
15 s |
/ |
|
Attention:
1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.
2) When the ideal amplification efficiency cannot be achieved, appropriately reduce the annealing temperature; When non-specific reactions occur, increase the annealing temperature.
3) If it is necessary to improve the efficiency of reaction amplification, the extension time can be appropriately increased.
4) This program is based on the ABI 7500 fluorescence quantitative PCR instrument as a reference setting. For melting curve analysis, please use the recommended program for the fluorescence quantitative PCR instrument used.
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