基本描述

英文名称

Phi29 DNA Polymerase

储存温度

-20°C储存,避免反复冻融

运输条件

超低温冰袋运输

产品介绍

Phi29 DNA Polymerase是从Bacillus subtilis噬菌体phi29中克隆出的DNA聚合酶，利用基因重组技术，由大肠杆菌表达。本产品具有高效的DNA连续合成能力及链置换能力，同时具有3'→5'外切酶校读功能，保真度高。本产品可应用于需要强置换和连续合成的复制反应及中温条件下高保真度复制，如质粒复制、全基因组扩增等。

P665534	Component	250 U	1250 U	Storage
P665534A	Phi29 DNA Polymerase, 10 U/μL	25 μL	125 μL	-20℃. Avoid freeze/thaw cycle.
P665534B	10×Phi29 Reaction Buffer	1 mL	1 mL	-20℃. Avoid freeze/thaw cycle.
P665534C	BSA, 10 mg/mL	200 μL	200 μL	-20℃. Avoid freeze/thaw cycle.

活性定义：

在30℃，10分钟内，将0.5 pmol的脱氧核苷酸掺入酸不溶性沉淀物所需的酶量定义为 1个活性单位（U）。

热失活:

65℃温育10 min即可失活。

质量控制：

经过多次柱纯化，SDS-PAGE检测其纯度大于95%；经检测无核酸内切酶活性，无宿主残留DNA。该酶缓冲液中含有还原剂DTT以便保证其最大酶活性，如果缓冲液不新鲜或经过反复冻融，使用前应添加4 mM的DTT。

应用实例：

利用Phi29 DNA聚合酶的特殊链置换和连续合成特性，可以大大简化用于测序的环状质粒制备过程。从细菌培养液中扩增质粒：取1 µl对数中后期新鲜培养物用于以下反应。从平板菌落中扩增质粒：挑取琼脂板上的菌落至10 µl（可变）的双蒸水中，混匀，取1 µl用于以下反应。扩增已纯化的环状质粒：质粒稀释至1 µg/ml，取1 µl用于以下反应。

1．样品加热变性及引物与质粒退火反应：加入以下组分，震荡混匀并短暂离心后，95℃ 加热3 min，然后置于冰上15 min。

2．扩增反应：在上述反应液中加入以下成分，震荡混匀并短暂离心后，30℃温育过夜。

3．65℃加热10 min，热失活Phi29 DNA Polymerase，终止反应。

4．扩增产物经稀释或纯化后即可用于测序。

Phi29 DNA Polymerase is a DNA polymerase cloned from the Bacillus subtilis bacteriophage phi29, expressed in Escherichia coli using gene recombination technology. This product has efficient DNA continuous synthesis ability and strand displacement ability, as well as 3 '→ 5' exonuclease reading function, with high fidelity. This product can be applied to replication reactions that require strong displacement and continuous synthesis, as well as high fidelity replication under medium temperature conditions, such as plasmid replication, whole genome amplification, etc.

P665534	Component	250 U	1250 U	Storage
P665534A	Phi29 DNA Polymerase, 10 U/μL	25 μL	125 μL	-20℃. Avoid freeze/thaw cycle.
P665534B	10×Phi29 Reaction Buffer	1 mL	1 mL	-20℃. Avoid freeze/thaw cycle.
P665534C	BSA, 10 mg/mL	200 μL	200 μL	-20℃. Avoid freeze/thaw cycle.

Activity definition:
The amount of enzyme required to add 0.5 pmol of deoxyribonucleotide to acid insoluble precipitate within 10 minutes at 30 ℃ is defined as 1 active unit (U).
Thermal deactivation:
Incubate at 65 ℃ for 10 minutes to inactivate.
Quality control:
After multiple column purifications, SDS-PAGE detected a purity of over 95%; After testing, there was no endonuclease activity and no residual host DNA. The enzyme buffer contains a reducing agent DTT to ensure its maximum enzyme activity. If the buffer is not fresh or has undergone repeated freeze-thaw cycles, 4 mM of DTT should be added before use.
Application examples:
By utilizing the special strand displacement and continuous synthesis properties of Phi29 DNA polymerase, the preparation process of circular plasmids for sequencing can be greatly simplified. Amplify plasmids from bacterial culture medium: Take 1 µ l logarithmic fresh medium for the following reaction. Amplify plasmids from agar plates: Take the colonies from the agar plates into 10 µ l (variable) double distilled water, mix well, and take 1 µ l for the following reaction. Amplify purified circular plasmid: Dilute the plasmid to 1 µ g/ml and take 1 µ l for the following reaction.
1. Sample heating denaturation and primer plasmid annealing reaction: Add the following components, shake well and centrifuge briefly, heat at 95 ℃ for 3 minutes, and then place on ice for 15 minutes.

2. Amplification reaction: Add the following components to the above reaction solution, shake well, centrifuge briefly, and incubate overnight at 30 ℃.

3.65 ℃ for 10 minutes to deactivate Phi29 DNA Polymerase and terminate the reaction.

4. The amplified product can be used for sequencing after dilution or purification.

AI解读

技术规格说明书

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

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溶液计算器

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货号 (SKU)	包装规格	是否现货	价格	数量
P665534-250U	250U	期货
P665534-1250U	1250U	期货