| Product Description | This product is a premixed system composed of GoldStar Best DNA Polymerase, Mg2+, dNTPs, PCR stabilizers and enhancers, with a concentration of 2 ×. It has the advantages of simple and fast operation, high sensitivity, strong specificity, and good stability, which can minimize human error and pollution. The GoldStar Best DNA Polymerase contained in this product is a chemically modified hot start high fidelity polymerase. This polymerase has 5 '-3' DNA polymerase activity, 5 '-3' exonuclease activity, and 3 '-5' exonuclease activity. Under normal PCR conditions, compared with GoldStar Taq DNA polymerase, it has excellent performance of high amplification efficiency and low mismatch rate. The chemically modified enzyme does not exhibit polymerase activity at room temperature, effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimers at room temperature. Enzyme activation requires incubation at 95 ℃ for 10 minutes, which can be integrated into existing PCR thermal cycling programs. The optimized buffer system maximizes the effectiveness of the enzyme, achieving high fidelity, specificity, amplification efficiency, and sensitivity for the target fragment. This product has been added with a dye (blue), and can be directly subjected to electrophoresis detection after the reaction is completed. Most PCR products obtained from amplification have an "A" base attached to the 3 'end, making them suitable for direct use in T/A cloning. Suitable for routine PCR reactions and gene cloning experiments that require high fidelity. | G665556 | Component | 1 mL | 5 mL | Storage | | G665556A | 2×GoldStar Best MasterMix (Dye) | 1 mL | 5×1 mL | -20℃. Avoid freeze/thaw cycle. | | G665556B | ddH₂O | 1 mL | 5×1 mL | -20℃. Avoid freeze/thaw cycle. |
Notes: 2×GoldStar Best MasterMix contains GoldStar Best DNA Polymerase, 3.4 mM MgCl₂ and 400 µM each dNTP. |
Quality control:
After testing, there was no exogenous nuclease activity; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes from multiple genomes; Storage at 2-8 ℃ for three months showed no significant change in activity. Usage:
The following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size. 1. PCR reaction system
| Reagent |
50 μlReaction system |
Final concentration |
| 2×GoldStar Best MasterMix（Dye） |
25 μl |
1× |
| Forward Primer，10 µM |
2 μl |
0.4 μM |
| Reverse Primer，10 µM |
2 μl |
0.4 μM |
| Template DNA |
<0.5 μg |
<0.5 μg/50 μl |
| ddH2O |
up to 50 μl |
/ |
|
Attention: The primer concentration should be between 0.1 and 1.0 as the final 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 reactions occur, the primer concentration can be reduced to optimize the reaction system. 2. PCR reaction conditions
| Step |
Temperature |
Time |
/ |
| Pre denaturation |
95℃ |
10 min |
/ |
| Denaturation |
94℃ |
30 s |
30-40 cycles |
| Anneal |
55-65℃ |
30 s |
30-40 cycles |
| Extend |
72℃ |
60 s |
30-40 cycles |
| Finally extended |
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, it should be appropriately reduced Low annealing temperature; 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, and the GoldStar Best DNA Polymerase contained in this product should be The amplification efficiency 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 the number of cycles is too high
Many, 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 must achieve enzyme activation under pre denaturation conditions of 95 ℃ and 10 minutes.
|
|---|
