| Product Description | GoldStar DNA Polymerase is a chemically modified and highly efficient Taq DNA Polymerase. The enzyme's activity is completely blocked at room temperature, making it inactive at low or room temperature. This effectively avoids 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. The unique buffer system makes the application of enzymes more extensive, enabling efficient amplification of high GC content, complex secondary structures, and low copy templates. Using this product for PCR amplification, the 3 'end of the PCR product contains an "A" base, which can be directly used for T/A cloning. This product has strong specificity and does not require gel recovery to remove impurities after PCR amplification. It can be directly used for downstream cloning or chip hybridization experiments. Mainly used for routine PCR, RT-PCR, Real time PCR, multiplex PCR, gene chip analysis, and SNP detection, especially suitable for PCR reactions that require high specificity. | G665840 | Component | 500 U | 2500 U | Storage | | G665840A | GoldStar DNA Polymerase, 5 U/μL | 100 μL | 5×100 μL | -20℃. Avoid freeze/thaw cycle. | | G665840B | 5×GoldStar PCR Buffer | 1.9 mL | 5×1.9 mL | -20℃. Avoid freeze/thaw cycle. |
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Attention: The 5 x GoldStar Taq PCR Buffer of this product contains 8.5mM magnesium ions. Unit Definition
Using activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxyribonucleotide into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.
Quality control
SDS-PAGE detection shows a purity greater than 99%; No exogenous nuclease activity detected; PCR method for detecting host free residual DNA; Can effectively amplify single copy genes in the human genome. Store at room temperature for one month without significant changes 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 μl Reaction system |
Final concentration |
| 5×GoldStar PCR Buffer |
10 μl |
1 × |
| dNTP Mix，10 mM each |
1 μl |
200 μM each |
| 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 |
| GoldStar DNA Polymerase，5 U/μl |
0.5 μl |
2.5 U/50 μl |
| ddH2O |
up to 50 μl |
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Attention: Primer concentration, please use final concentration of 0.1-1.0 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. PCR reaction conditions
| Step |
Temperature |
Time |
/ |
| Pre denaturation |
95℃ |
10 min |
/ |
| Denaturation |
95℃ |
30 s |
30-40 cycles |
| Annealing |
55-65℃ |
30 s |
30-40 cycles |
| Extension |
72℃ |
60 s |
30-40 cycles |
| Final extension |
72℃ |
5 min |
/ |
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Attention:
1) In general experiments, the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the annealing time is generally 30-60 seconds. If 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 Taq 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 must achieve enzyme activation under pre denaturation conditions of 95 ℃ and 10 minutes.
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