| Product Description | FastSYBR 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 Fast 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. The Fast Taq DNA Polymerase contained in this product can effectively reduce non-specific amplification caused by non-specific binding of primers and templates or primer dimers at room temperature. Enzyme activation only requires incubation at 95 ℃ for 20 seconds. The entire PCR reaction process can save about 40 minutes compared to ordinary reactions, greatly reducing the reaction time of PCR. The unique combination of PCR buffer system and hot start enzyme effectively inhibits the production of non-specific products and significantly improves the amplification efficiency of PCR. This product has a wide range of applications and is suitable for both regular and rapid quantitative PCR programs. ROX dye is used to correct the fluorescence signal error between wells in quantitative PCR instruments, and is generally used in Real Time PCR amplification instruments from companies such as ABI and Stratagene. The excitation optical systems of different instruments vary, so the concentration of ROX dye must be matched with the corresponding fluorescence quantitative PCR instrument.
Instruments that do not require ROX calibration: Roche LightCycle 480, Roche LightCylinder 96, Bio rad iCylinder iQ, iQ5, CFX96, etc.
Instruments that require Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio ® 3 System, QuantStudio ® 5 System, QuantStudio ® 6 Flex System, QuantStudio ® 7 Flex System, ViiA 7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, etc.
Instruments that require High ROX calibration include ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.
| Component |
F665884 5 ml |
| 2×FastSYBR Mixture |
5×1 ml |
| 50×Low ROX |
/ |
| 50×High ROX |
/ |
| ddH2O |
5×1 ml |
|
Matters needing attention
1. Before use, please gently mix upside down to avoid foaming, and use after briefly centrifugation.
2. This product contains the fluorescent dye SYBR Green I. When storing this product or preparing PCR reaction solution, strong light exposure should be avoided.
3. Avoid repeated freezing and thawing of this product, as repeated freezing and thawing may cause a decrease in product performance. This product can be stored for a long time at -20 ℃, away from light. If frequent use is required in the short term, it can be stored at 2-8 ℃.
4. This product cannot be used for probe based fluorescence quantitative PCR.
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×FastSYBR 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⁾ |
/ |
50×Low ROX or High ROX
（optional）³⁾ |
1 μL |
1 × |
| ddH2O |
up to 50 μl |
/ |
|
Attention:
1) Typically, the primer concentration is 0.2 μ M can achieve good results, ranging from 0.1 to 1.0 μ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.
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 excitation optical systems of different instruments vary, and 50 x Low ROX or 50 x High ROX can be added according to the instrument used for fluorescence quantification
2. PCR reaction conditions
| Step |
Temperature |
Time |
/ |
| Pre denaturation |
95℃ |
20 s ¹⁾ |
/ |
| Denaturation |
95℃ |
3 s |
35-40 cycles |
| Annealing/Extension ² ⁾ |
60℃ |
30 s |
35-40 cycles |
| Analysis of Fusion Curve ³ ⁾ |
/ |
/ |
/ |
| / |
95℃ |
15 s |
/ |
| / |
60℃ |
1 min |
/ |
| / |
95℃ |
15 s |
/ |
| / |
60℃ |
15 s |
/ |
|
Attention:
1) The enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 20 seconds. Under these conditions, most templates can perform well in de chaining. For templates with high GC content and complex secondary structures, the pre denaturation time can be extended to 1 minute to fully unwind the starting template. If the high-temperature treatment time is too long, it will affect the enzyme activity. The optimal pre denaturation time can be determined based on the template situation.
2) It is recommended to use a two-step PCR reaction program, and the annealing temperature should be set at 60-64 ℃ as a reference range. When non-specific reactions occur, the annealing temperature can be increased. 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 should be set within the range of 56 ℃ -64 ℃ as a reference.
3) Please set the fusion curve analysis according to the recommended program of the fluorescence quantitative PCR instrument used. This program is based on the ABI 7500 fluorescence quantitative PCR instrument as a reference setting.
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