| Product Description | GoldStar Probe Mixture is a premixed system specifically designed for real-time fluorescence quantitative PCR using probe methods (TaqMan, Molecular Beacon, etc.), with a concentration of 2 x, containing GoldStar Taq DNA Polymerase, PCR Buffer, dNTPs, and Mg2+. The operation is simple and convenient. Mainly used for detecting genomic DNA target sequences and RNA reverse transcription cDNA target sequences, such as gene expression analysis, copy number analysis, SNP genotype analysis, etc., suitable for fluorescence quantification using different types of probe methods. The GoldStar Taq DNA Polymerase contained in this product is a chemically modified, novel and highly efficient hot start enzyme. It 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. The enzyme activation requires incubation at 95 ℃ for 10 minutes. The unique combination of PCR buffer system and hot start enzyme significantly improves the amplification efficiency of PCR, with stronger fluorescence signal and higher sensitivity, which can detect single copy templates. By using this product, a wider linear range can be obtained, resulting in more accurate quantification of the target gene. Suitable for all fluorescence quantitative PCR instruments that do not require ROX as a calibration dye.
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 (CW0932): Roche LightCycle 480, Roche LightCyler 96, Bio rad iCyler iQ, iQ5, CFX96, etc.
Instrument requiring Low ROX calibration (CW2625): 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 (CW2626): ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc. | G665832 | Component | 5 mL | Storage | | G665832A | 2×GoldStar Probe Mixture | 5×1 mL
| -20℃. Avoid freeze/thaw cycle. | | G665832B | ddH2O | 5×1 mL
| -20℃. Avoid freeze/thaw cycle. |
|
Notes:
1. Before use, please gently mix upside down to avoid foaming, and use after briefly centrifugation.
2. 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 ℃.
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×GoldStar Probe Mixture |
25 μl |
1 × |
| Forward Primer，10 µM |
1 μl |
0.2 μM¹⁾ |
| Reverse Primer，10 µM |
1 μl |
0.2 μM¹⁾ |
| Probe，10 µM |
1 μl |
0.2 μM²⁾ |
| Template DNA |
2 μl³⁾ |
/ |
| 50×Low ROX or High ROX（optional）⁴⁾ |
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.
2) The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance. Please refer to the instrument manual or the specific usage requirements of each fluorescent probe for concentration adjustment during actual use.
3) 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.
4) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added. 2. PCR reaction program
Attention! The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes!
Two step PCR
| Step |
Temperature |
Time |
/ |
| Pre denaturation |
95℃ |
10 min¹⁾ |
/ |
| Denaturation |
95℃ |
15 s |
35-40 cycles |
| Annealing/Extension ²⁾ |
60℃ |
1 min |
35-40 cycles |
|
Attention:
1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.
2) It is recommended to use a two-step PCR reaction program. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification can be attempted. The annealing temperature should be set within the range of 56 ℃ -64 ℃ as a reference.
|
|---|
