Super Pfx DNA Polymerase是一种快速、高扩增效率的高保真DNA聚合酶，该聚
合酶具有5′-3′DNA聚合酶活性和3′-5′外切酶活性。该酶经其他高保真酶改造而来，扩增
能力强、扩增速度快，保真度高，克服了普通Pfu酶扩增能力差、产量低和扩增速度慢
的缺陷，极大地缩短了反应时间。本品可用于长片段扩增及其他各种复杂模板的扩
增，扩增得到的PCR产物的3′端不带有“A”碱基，可直接克隆于平末端载体中，如需进
行T/A克隆，需在PCR产物末端添加“A”后进行克隆。本产品适用于基因克隆、基因定
点突变、SNP等扩增实验。
| S665588 | Component | 100 U | 500 U | Storage | | S665588A | Super Pfx DNA Polymerase, 2 U/μL | 50 μL | 250 μL | -20℃. Avoid freeze/thaw cycle. | | S665588B | 2×Super Pfx Buffer | 2×1.25 mL | 7×1.8 mL | -20℃. Avoid freeze/thaw cycle. | | S665588C | dNTP Mix, 10mM each | 150 μL | 750 μL | -20℃. Avoid freeze/thaw cycle. |
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活性定义
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
经过多次柱纯化，SDS-PAGE检测其纯度大于98%；经检测无外源核酸酶活性；
室温存放一个月，无明显活性改变。
使用方法
以下举例为常规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
所有操作请在冰上进行，各组分解冻后请充分混匀，用完之后请及时放回-20℃保存。
2. PCR 反应条件
注意：
1）优先使用三步法扩增，三步法无法扩增目的产物或引物Tm值大于68°C，请尝试两步法。
2）变性：简单模板的预变性98℃，30s-1min，对于复杂的模板，预变性时间可延长至3min。
3）退火：一般实验中退火温度比引物的Tm值低3-5℃，如无法得到理想的扩增效率时，应梯度改变退
火温度，进行优化；发生非特异性反应时，适当提高退火温度。
4）延伸：延伸时间应根据所扩增片段的长度和模板复杂程度设定，本产品扩增效率为3-5 kb/min，对于
长片段及复杂性高的模板建议2-4kb/min。
5）循环次数：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，循环次数太
多，错配机率会增加，所以在保证产物得率的前提下应尽量减少循环次数。
Super Pfx DNA Polymerase is a fast and highly efficient high fidelity DNA polymerase with 5 ′ -3 ′ DNA polymerase activity and 3 ′ -5 ′ exonuclease activity. This enzyme is modified from other high fidelity enzymes, with strong amplification ability, fast amplification speed, and high fidelity, overcoming the shortcomings of poor amplification ability, low yield, and slow amplification speed of ordinary Pfu enzymes, greatly shortening the reaction time. This product can be used for long fragment amplification and other complex template amplification. The 3 'end of the PCR product obtained from amplification does not contain an "A" base and can be directly cloned into a flat end vector. If T/A cloning is required, "A" needs to be added to the end of the PCR product before cloning. This product is suitable for gene cloning, gene directed mutagenesis, SNP amplification experiments, etc.
| S665588 | Component | 100 U | 500 U | Storage | | S665588A | Super Pfx DNA Polymerase, 2 U/μL | 50 μL | 250 μL | -20℃. Avoid freeze/thaw cycle. | | S665588B | 2×Super Pfx Buffer | 2×1.25 mL | 7×1.8 mL | -20℃. Avoid freeze/thaw cycle. | | S665588C | dNTP Mix, 10mM each | 150 μL | 750 μL | -20℃. Avoid freeze/thaw cycle. |
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Activity definition
The amount of enzyme required to add 10 nmoL deoxyribonucleotides to acidic insoluble substances within 30 minutes at 74 ℃ is defined as 1 active unit (U).
Quality control
After multiple column purifications, SDS-PAGE detected a purity of over 98%; No exogenous nuclease activity detected; Store at room temperature for one month without significant changes 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
All operations should be carried out on ice. After each group is decomposed and frozen, please mix thoroughly. After use, please put it back at -20 ℃ for storage in a timely manner.
Reagent | 50 μLReaction system | final concentration | 2×Super Pfx Buffer | 25 μL | 1× | dNTP Mix，10 mM each | 1.5-2.5 μL | 300-500 μM each | Forward Primer，10 μM | 2 μL | 0.4 μM | Reverse Primer，10 μM | 2 μL | 0.4 μM | TemplateDNA appropriate amount | appropriate amount | <500 ng/50 μL | Super Pfx DNA Polymerase | 0.5-0.75 μL | 1-1.5 U/50 μL | ddH2 O | up to 50 μL | |
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2.
PCR reaction conditions
step | temperature | time | | Pre denaturation | 98°C | 30 s-3 min | | Degeneration regression | 98°C | 10-30 s | 25-35 cycles | Fire extension | Determine based on primer Tm | 15-30 s | 25-35 cycles | Final extension | 72°C | 3-5 kb/min | 25-35 cycles | extend | 72°C | 5 min | |
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Attention:
1) Priority should be given to using the three-step method for amplification. If the three-step method cannot amplify the target product or the Tm value of the primer is greater than 68 ° C, please try the two-step method.
2) Denaturation: Pre denaturation of simple templates at 98 ℃ for 30 seconds to 1 minute. For complex templates, the pre denaturation time can be extended to 3 minutes.
3) Annealing: In general experiments, the annealing temperature is 3-5 ℃ lower than the Tm value of the primer. If the ideal amplification efficiency cannot be achieved, the annealing temperature should be gradually changed for optimization; When non-specific reactions occur, increase the annealing temperature appropriately.
4) Extension: The extension time should be set according to the length of the amplified fragment and the complexity of the template. The amplification efficiency of this product is 3-5 kb/min, and it is recommended to 2-4 kb/min for long fragments and high complexity templates.
5) Cycle count: The number of cycles can be set based on the downstream application of the amplification product. If there are too few cycles, insufficient amplification, or too many cycles, the probability of mismatch will increase. Therefore, while ensuring product yield, the number of cycles should be minimized as much as possible.
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