68kDa (Reducing)
Store at -25 ~ -15℃ for 2 years
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. Prepare the reaction system on the ice bath with reference to the table below.
Reagent | Volume | Final Concentration |
10X Reaction Buffer | 2 ul | 1 X |
dNTP (2.5 mM each) | 1 ul | 125 uM |
Random Hexamer Primers (100μM) | 1 ul | 5 mM |
Template DNA (≥1 ng) | x ul | - |
Nuclease-free Water | (15-x) ul | - |
Total Volume | 19 ul | - |
*
phi29 DNA Polymerase has a strong 3'→5' exonuclease activity. If the amplification effect is not good, it is recommended to reduce the amount of enzyme in the reaction system to 0.5μl or 0.25μl.
2. Predegeneration of template DNA: The reaction system was incubated in a PCR apparatus at 95 ℃ for 5min, and quickly placed in an ice bath for 2min or longer.
3. Isothermal amplification reaction: 1μl phi29 DNA Polymerase was added into the cooled reaction system and incubated at 30℃ for 2-16h. Incubation is usually sufficient for 2h, and if a larger amount of amplified product is desired, the incubation time can be extended to 16h. It is recommended to use a constant temperature water bath for the reaction. If using a hot cap PCR apparatus, adjust the hot cap temperature to 40 ° C to avoid enzyme inactivation.
4. Termination reaction: incubation at 65℃ for 10 min.
5. Detection of amplification products: The amplification products were subjected to agarose gel electrophoresis to detect the amplification effect.
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.
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;
2μg (R: reducing condition, N: non-reducing condition).
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