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分子别名
phi29 DNA Polymerase、DNA polymerase表达宿主
E.coli分子量
68kDa (Reducing)
纯度
≥95% by SDS-PAGE活性
10U/μl标签
His Tag缓冲体系
100 mM KCl、10 mM Tris-HCl、0.1 mM EDTA、1 mM DTT、0.5% Tween® 20、50% Glycerol、0.5% NP40、pH 7.4 @ 25°C储存条件
Store at -25 ~ -15℃ for 2 years
文献引用
1. Tadas P, Gediminas A , Rasa S ,et al. In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self-replication technique[J].Protein Engineering Design & Selection, 2016(12):617-628.
2. Margarita S, Isabel H, Redrejo-Rodríguez Modesto,et al. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication[J].Frontiers in Molecular Bioences, 2016, 3:37.
phi29 DNA polymerase is a DNA polymerase cloned from Bacillus subtilis phage phi29 (Φ29) (1). On the one hand, the enzyme has excellent strand replacement and sustained synthesis capabilities, enabling the unstranding and replication of complex DNA structures and isothermal DNA polymerization reactions in vitro that do not depend on thermal cycling. On the other hand, the enzyme possesses 3´→5´ nucleic acid exonuclease proofreading activity.
10U/μl phi29 DNA Polymerase、100 mM KCl、10 mM Tris-HCl、0.1 mM EDTA、1 mM DTT、0.5% Tween® 20、50% Glycerol、0.5% NP40、pH 7.4 @ 25°C
Reaction buffer: 50 mM Tris-HCl、10 mM MgCl2、10 mM (NH4)2SO4、4 mM DTT、(pH 7.5 @ 25°C)
1. The active reducing agent in the reaction buffer is critical for this enzyme. Although the reaction buffer supplied with the enzyme contains DTT, in order to ensure maximum activity, 4 mM DTT should be added when using buffers that have been stored for long periods of time or buffers that have been repeatedly freeze-thawed.
2.Reaction temperatures above 65°C are not recommended.
3.The enzyme does not have 5´→3´ nucleic acid exonuclease activity.
- One unit refers to the amount of enzyme required to catalyze the incorporation of 0.5 pmol of dNTP into an acid-insoluble material in 10 minutes at 30 °C.
生物活性
Comparison of the amplification effect of phi29 DNA Polymerase developed by UA and similar products of foreign N company on pCMV-N-DsRed (4959 bp). The above diagram shows the effect of incubation at 30℃ for 16h. The Reaction system was 20μl: 2μl 10X Reaction Buffer, 1μl Random Hexamer Primer (100 μM), 1μl dNTP (2.5 mM each), and 1μl 5kb circular plasmid DNA (pCMV-N-DsRed, 10 ng/μl), 14μl Nuclease-free Water, incubated at 95℃ for 5 min, and then added 1μl phi29 DNA Polymerase (10 U/ul) after 2 min in ice bath. Add 4μl 6X DNA Loading buffer, take 10μl reaction product, and use 1% agarose gel electrophoresis to detect the amplification effect.
1. Negative control -1 (only the template pCMV-N-DsRed was not added, but the normal amount of phi29 DNA Polymerase was added);
2. Negative control -2 (only phi29 DNA Polymerase was not added);
3. UA phi29 DNA Polymerase 10U;
4. Competing product N 10U;
电泳
2μg (R: reducing condition, N: non-reducing condition).
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