产品内容
| F665904 | Component | 500 U | 2500 U | Storage | | F665904A | FastStar DNA Polymerase, 5 U/μL | 100 μL | 5×100 μL | -20℃. Avoid freeze/thaw cycle. | | F665904B | 10×PCR Buffer | 1.8 mL | 5×1.8 mL | -20℃. Avoid freeze/thaw cycle. |
Notes: 10×PCR Buffer contains 15mM Mg2+. |
产品简介
FastStar DNA Polymerase是抗Taq酶单克隆抗体和CW0680 Taq DNA Polymerase 的混合制品，适用于Hot Start PCR。使用Taq酶抗体进行PCR扩增时，高温变性前由于Taq酶抗体与Taq酶结合抑制DNA聚合酶活性，能够在低温条件下有效抑制引物的非特异性退火及引物二聚体引起的非特异性扩增。Taq酶抗体在PCR反应最初的DNA变性步骤中变性，DNA聚合酶活性恢复，达到热启动效果。使用本品无需特殊地对Taq酶抗体失活处理，可以在常规PCR反应条件下使用。
FastStar DNA Polymerase具有5′→3′ DNA聚合酶活性和5′→3′外切酶活性，无3′→5′外切酶活性，酶延伸速度2 kb/min，可以扩增长度达5 kb的片段。扩增得到的PCR 产物3′端附有一个“A”碱基，因此可直接用于T/A克隆。本产品具有延伸速度快、扩增效率高的特点，主要适用于PCR法扩增DNA片段、DNA序列测定等实验。
活性定义
用活性化的大马哈鱼精子DNA作为模板/引物，在74°C，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99%；经检测无外源核酸酶活性；
PCR方法检测无宿主残余DNA；能有效地扩增人基因组中的单拷贝基因。
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 |
| 10× PCR Buffer |
5 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 |
| FastStar DNA Polymerase,5U/μl |
0.25-0.5 μl |
1.25-2.5 U/50 μl |
| ddH₂O |
up to 50 μl |
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注意：可以在室温下配制反应液，须将试剂置于冰上。
2. PCR反应条件
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℃，无法得到理想的扩增效率时，适当降 低退火温度。
2） 延伸时间应根据所扩增片段大小设定。
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。
Product content
| F665904 | Component | 500 U | 2500 U | Storage | | F665904A | FastStar DNA Polymerase, 5 U/μL | 100 μL | 5×100 μL | -20℃. Avoid freeze/thaw cycle. | | F665904B | 10×PCR Buffer | 1.8 mL | 5×1.8 mL | -20℃. Avoid freeze/thaw cycle. |
Notes: 10×PCR Buffer contains 15mM Mg2+. |
Product Introduction
FastStar DNA Polymerase is a mixture of anti-Taq enzyme monoclonal antibody and CW0680 Taq DNA Polymerase for Hot Start PCR.When using Taq enzyme antibody for PCR amplification, Taq enzyme antibody binds to Taq enzyme to inhibit the DNA polymerase activity prior to high temperature denaturation, and effectively inhibits non-specific annealing of primers and non-specific amplification caused by primer dimerization at low temperatures. It can effectively inhibit the non-specific annealing of primers and non-specific amplification caused by primer dimer under low temperature conditions.Taqase antibody denatures the DNA in the initial DNA denaturation step of the PCR reaction, and DNA polymerase activity is restored to achieve the effect of hot start. No special inactivation of Taq enzyme antibody is required for the use of this product, and it can be used under conventional PCR reaction conditions.
FastStar DNA Polymerase with 5′→3′ DNA polymerase activity and 5′→3′ exonuclease activity, no 3′
→With 5′ exonuclease activity and an enzyme extension rate of 2kb/min, fragments up to 5kb in length can be amplified. The amplified PCR product has an "A" base at the 3′ end, so it can be directly used for T/A cloning. This product is characterized by fast extension speed and high amplification efficiency, and is mainly suitable for PCR amplification of DNA fragments, DNA sequence determination and other experiments.
Active Definition
Using activated salmon sperm DNA as template/primer, the amount of enzyme required to dope 10 nmol of deoxyribonucleotide into acid-insoluble material was defined as 1 activity unit (U) at 74°C for 30 min.
quality control
After several column purifications, its purity was greater than 99% by SDS-PAGE; no exogenous nuclease activity was detected;
PCR method detects no host residual DNA; efficiently amplifies single-copy genes in the human genome.
Usage
The following example shows the PCR reaction system and reaction conditions for amplifying a 1kb fragment using human genomic DNA as a template, which should be improved and optimized according to the template, primer structure and size of the target fragment in actual operation.
| Reagent | 50 μl Reaction system | final concentration | | 10× PCR Buffer | 5 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 | | FastStar DNA Polymerase,5U/μl | 0.25-0.5 μl | 1.25-2.5 U/50 μl | | ddH₂O | up to 50 μl |
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Note: The reaction solution can be prepared at room temperature; the reagents must be placed on ice.
2.PCR reaction conditions
Attention:
1) In general, the annealing temperature in the experiment is 5°C lower than the melting temperature Tm of the amplification primer, and when the ideal amplification efficiency cannot be obtained, the annealing temperature is appropriately lowered.
2) The extension time should be set according to the size of the amplified fragment.
3) The number of cycles can be set according to the downstream application of the amplification product . If the number of cycles is too low, the amplification is insufficient; if the number of cycles is too high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield.
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