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产品说明
Anstart PCR Mix 为预先混合的Anstart Taq DNA聚合酶、反应缓冲液、Mg2+（终浓度2mM）和dNTP的浓缩溶液。DNA模版和引物简单添加即可开始PCR反应。常规PCR扩增的一致性和效率得以优化。
产品用途
热启动法进行PCR 扩增。
使用方法
1.PCR 反应体系的设置
a.溶解并混匀 PCR 反应所需的各种溶液，并放置于冰浴上或冰盒内。建议反应 PCR 液体分装使用， 避免反复冻融。
b.参考下表设置 PCR 反应，建议 PCR 反应体系的配置在冰浴或在冰盒上进行：
试剂 | 体积 | 终浓度 |
Anstart PCR Mix（2×） | 25μl |
|
Primer-probe Mix | 3μl | — |
模板 | 10μl ※ | — |
超纯水 | Up to 50μl | — |
总体积 | 50μl | — |
※对于不同类型的模板在 50μl 反应体积中推荐用量如下：
哺乳动物基因组 DNA：0.1-1μg
大肠杆菌基因组 DNA：10-100ng
质粒 DNA：0.1-10ng
过多的模板 DNA 容易导致非特异性的 PCR 产物
c.用移液器轻轻吹打混匀或轻微 Vortex 混匀，室温离心数秒，使液体积聚于管底。
d.各设置好的 PCR 反应管置于 PCR 仪上，开始 PCR 反应。
2.PCR 反应参数的设置以扩增 1Kb 目的片段为例
步骤 | 温度 | 时间 | 循环数 |
预变性 | 95℃ | 2.5min | 1 |
变性 | 94℃ | 30s |
25-35 |
退火 | 55℃ | 30s | |
延伸 | 72℃ | 1min | |
最后延伸 | 72℃ | 10min | 1 |
a.PCR 反应的设置需根据模板、引物、PCR 产物的长度和 GC 含量等条件的不同设定不同的PCR
反应条件包括温度、时间和循环数等。
b.STEP4(延伸)的时间设置需根据 PCR 产物的长度进行设置，通常每 kb 产物的延伸时间为 1min。例如 PCR 产物的长度为 1kb，则延伸时间可以设置为 1min，PCR 产物的长度为 2kb，则延伸时间可以设置为 2min，以此类推。
c .对于初次进行的PCR，为尽量确保可以扩增出预期的 PCR 产物，可以把循环数设置为 35。对于需进行半定量或定量的PCR 反应循环数一定要进行适当优化，使PCR 反应达到平台期。
Product manual
Anstart PCR Mix is a pre-mixed concentrated solution of Anstart Taq DNA polymerase, reaction buffer, Mg2+ (final concentration 2mM) and dNTP. Simply add DNA template and primers to start the PCR reaction. The consistency and efficiency of conventional PCR amplification are optimized.
Product Usage
Hot start method for PCR amplification.
Instructions
1. PCR reaction system settings
a. Dissolve and mix the various solutions required for the PCR reaction, and place them on an ice bath or in an ice box. It is recommended that the reaction PCR liquid be used in aliquots to avoid repeated freezing and thawing.
b. Refer to the following table to set up the PCR reaction. It is recommended to configure the PCR reaction system in an ice bath or on an ice box:
Reagent | Volume | Final concentration |
Anstart PCR Mix（2×） | 25μl |
|
Primer-probe Mix | 3μl | — |
Template | 10μl ※ | — |
Ultra-pure water | Up to 50μl | — |
Total capacity | 50μl | — |
※For different types of templates, the recommended dosage in 50μl reaction volume is as follows:
Mammalian genomic DNA: 0.1-1μg
Escherichia coli genomic DNA: 10-100ng
Plasmid DNA: 0.1-10ng
Too much template DNA can easily lead to non-specific PCR products
c. Use a pipette to mix gently or gently Vortex to mix, and centrifuge for a few seconds at room temperature to allow the liquid to accumulate at the bottom of the tube.
d. Place each set of PCR reaction tubes on the PCR machine to start the PCR reaction.
2. The setting of PCR reaction parameters takes the amplification of 1Kb target fragment as an example
Step | Temperature | Time | Number of cycles |
Predenaturation | 95℃ | 2.5min | 1 |
Transsexual | 94℃ | 30s |
25-35 |
Annealing | 55℃ | 30s | |
Extend | 72℃ | 1min | |
Last extension | 72℃ | 10min | 1 |
a. PCR reaction settings need to be set according to different conditions such as template, primers, PCR product length and GC content, etc.
The reaction conditions include temperature, time and number of cycles.
b. The time setting of STEP4 (extension) needs to be set according to the length of the PCR product, usually the extension time per kb product is 1 min. For example, if the length of the PCR product is 1kb, the extension time can be set to 1min, and the length of the PCR product is 2kb, then the extension time can be set to 2min, and so on.
c. For the initial PCR, in order to ensure that the expected PCR product can be amplified as much as possible, the number of cycles can be set to 35. For semi-quantitative or quantitative PCR reaction cycles, the number of cycles must be properly optimized to make the PCR reaction reach a plateau.
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