| Product Description | Product content:
| G666077 | Component | 5 mL | Storage | | G666077A | 2×Gold Multiplex PCR Mix | 5×1 mL | -20°C. Avoid freeze/thaw cycle.
| | G666077B | ddH₂O | 5×1 mL | -20°C. Avoid freeze/thaw cycle. |
|
Product Introduction:
Gold Multiplex PCR Mix is a premixed system composed of GoldStar DNA Polymerase, Mg2+, dNTPs, as well as PCR stabilizers and enhancers. Using this product does not require a complicated optimization process for PCR reaction conditions, and multiple PCR reactions can be easily carried out with simple condition exploration. The GoldStar DNA Polymerase contained in this product is a chemically modified hot start enzyme that can effectively reduce non-specific amplification caused by primer mismatch in the early stages of PCR reactions. The activation of the enzyme needs to be incubated at 95 ° C for 10 minutes. This enzyme, in combination with PCR enhancers that can enhance reaction specificity and a unique buffer system, allows all primers in the reaction system to be effectively extended without the need for additional optimization. This MasterMix also includes GC Enhancer, which helps achieve efficient amplification of "difficult" templates, such as those with high GC content. Gold Multiplex PCR Mix is suitable for various types of multiplex PCR reactions, such as microsatellite analysis, genotyping, and SNP detection. quality control：
After testing, there was no exogenous nuclease activity; PCR method for detecting residual DNA without host; Storage at 2-8 ℃ for three months showed no significant change in activity. 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 μlReaction system |
final concentration
|
| 2×Gold Multiplex PCR Mix |
25 μl |
1× |
| Primer Mix，10 µM each |
1 μl |
0.2 μM |
| Template DNA |
appropriate amount
|
/ |
| ddH2O |
up to 50 μl |
/ |
|
Attention: When designing primers, try to minimize the difference in Tm between each primer, and control the difference within 5 ℃ as much as possible. Please use the final concentration of 0.05-0.2 for each primer concentration 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 amplification occurs, the primer concentration can be reduced to optimize the reaction system. 2. PCR reaction conditions
| step |
temperature |
time |
/ |
| Pre denaturation |
95℃ |
10 min |
/ |
| denaturation |
95℃ |
30 s |
30-40cycle |
| anneal |
55-65℃ |
30 s |
30-40cycle |
| extension |
72℃ |
1 kb/min |
30-40cycle |
| Final extension |
72℃ |
5 min |
/ | |
Attention:
1) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.
2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of GoldStar DNA Polymerase contained in this product is 1-2 kb/min.
3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible.
4) This product achieves enzyme activation under pre denaturation conditions of 95 ° C for 10 minutes.
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