GoldStar DNA Polymerase是一种经化学修饰、全新高效的Taq DNA Polymerase，
在常温下酶的活性被完全封闭，使得该酶在低温或常温下没有活性，从而有效避免在
常温条件下由引物和模板非特异性结合或引物二聚体而产生的非特异性扩增，酶的激活
须在95℃下孵育10分钟。独特的缓冲体系使酶的应用更为广泛，使GC含量高、二级
结构复杂以及低拷贝的模板，均能高效扩增。用本品进行PCR扩增，PCR产物3'末端
附有一个“A”碱基，可直接用于T/A 克隆。本产品特异性强，PCR扩增后无需胶回收去
除杂带，可直接用于下游克隆或芯片杂交等实验。主要应用于常规的PCR、RT-PCR、
Real-time PCR、多重PCR、基因芯片分析和SNP检测，特别适用于对特异性要求较高
的PCR反应。
| 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. |
|
注意：本产品的5×GoldStar Taq PCR Buffer中含有8.5mM镁离子。
单位定义
用活性化的大马哈鱼精子DNA作为模板/引物，在74℃，30分钟内，将10 nmol脱氧
核苷酸掺入到酸性不溶物质所需的酶量定义为1个活性单位（U）。
质量控制
SDS-PAGE检测纯度大于99%；经检测无外源核酸酶活性；PCR方法检测无宿主
残留DNA；能有效扩增人基因组中的单拷贝基因。室温存放一个月，无明显活性改变。
使用方法
以下举例为以人基因组DNA为模板，扩增1 kb的片段的PCR反应体系和反应条件，实
际操作中应根据模板、引物结构和目的片段大小不同进行相应的改进和优化。
1. PCR反应体系
| 试剂 |
50 μl反应体系 |
终浓度 |
| 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 |
/ |
|
注意：引物浓度，请以终浓度0.1-1.0 μM作为设定范围的参考。扩增效率不高的情况下，可提
高引物的浓度；发生非特异性反应时，可降低引物浓度，由此优化反应体系。
2. PCR反应条件
| 步骤 |
温度 |
时间 |
/ |
| 预变性 |
95℃ |
10 min |
/ |
| 变性 |
95℃ |
30 s |
30-40 个循环 |
| 退火 |
55-65℃ |
30 s |
30-40 个循环 |
| 延伸 |
72℃ |
60 s |
30-40 个循环 |
| 终延伸 |
72℃ |
5 min |
/ |
|
注意：
1）一般实验中退火温度比扩增引物的熔解温度Tm低5℃，退火时间一般为30-60 s，无法得到理
想的扩增效率时，适当降低退火温度；发生非特异性反应时，提高退火温度，由此优化反应条
件。
2）延伸时间应根据所扩增片段大小设定，本产品中所包含的GoldStar Taq DNA Polymerase的扩
增效率为1-2 kb/min。
3）可根据扩增产物的下游应用设定循环数。如果循环次数太少，扩增量不足；如果循环次数太
多，错配机率会增加，非特异性背景严重。所以在保证产物得率的前提下应尽量减少循环次数。
4）本产品须在预变性95℃，10 min条件下实现酶的活化。
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. |
|
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 |
/ |
|
|
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 |
/ |
|
|
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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