phi29 DNA Polymerase,UA BIOSCIENCE_specification/price/image

Product Detail
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Product Specification

Synonyms	phi29 DNA Polymerase、DNA polymerase
Expression System	E.coli
Molecular Weight	68kDa (Reducing)
Purity	≥95% by SDS-PAGE
Tag	His Tag
Storage Buffer	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
Stability & Storage	Store at -25 ~ -15℃ for 2 years
Reference	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.

Background

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.

Components

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)

Protocol

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.

Guidelines

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.

Unit Definition

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.

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.