Phi29 DNA Polymerase, 300 U

Product Information

Product Introduction

This product, phi29 DNA polymerase, is derived from the thermophilic Bacillus phage phi29 and is recombinantly expressed in Escherichia coli. It has DNA chain displacement activity and strong DNA continuous synthesis ability (can achieve continuous polymerization extension of more than 70 kb in length), 3'-5' exonuclease activity, and has strong fidelity. It is mainly used for rolling circle isothermal amplification (RCA), genome amplification sequencing, etc.

Source : DNA Polymerase gene from Bacillus subtilis bacteriophage phi29, purified by recombinant expression in Escherichia coli.

Enzyme activity definition: The amount of enzyme required to incorporate 0.5 pmol dNTP into the acid-insoluble precipitate within 10 min at 30°C is defined as one unit of enzyme activity.

Purity and concentration: Purity detected by SDS-PAGE 95%; residual endogenous nucleic acid <10 ^-1  pg/μL (qPCR detection); 10 U/μL.

Inactivation or inhibition: Heat at 65°C for 10 min to inactivate.

Enzyme Storage Buffer: 10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% Tween 20, 50% Glycerol, pH 7.4.

10×phi29 Reaction Buffer：200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.8。

Figure 1. The effect of phi29 DNA Polymerase catalyzing plasmid (A) and 293 cell genome (B) DNA amplification. The reaction system is 20 μL, the template in Figure A is pUC19 plasmid, the template in Figure B is 293 cell genome, and the primers are all random primers. The whole system was incubated at 95 for 5 min, and after ice bathing for 2 min, 1 μL of phi29 DNA Polymerase with different dilution ratios (8, 4, 2, 1 times) was added, corresponding to No. 3, 4, 5, and 6, respectively. No phi29 DNA Polymerase was added to No. 1, and 1 μL of phi29 DNA Polymerase was added to No. 2, but no template was added. The system was incubated at 30 for 2 h.

Storage and transportation

Transported with wet ice; stored at -20, valid for 12 months.

composition

Procedure

1. Prepare the reaction system according to the following table:

2. Pre-denaturation of template DNA: Incubate the above reaction system at 95°C for 5 min and quickly place on ice for 2 min.

3. Constant temperature amplification reaction: Add 1 μL phi29 DNA Polymerase to the cooled reaction system and incubate at 30°C for 2-16 hours. (Generally, incubate for 2 hours. If you want to obtain more products, you can extend the time to 16 hours.)

4. End the reaction: incubate at 65°C for 10 min.

5. The amplified product can be detected by agarose gel electrophoresis.

Precautions

1. For the application of phi29 DNA Polymerase in rolling circle amplification (RCA), please refer to the relevant usage methods.

2. Enzyme products should be operated on an ice bath to avoid leaving the enzyme at room temperature for a long time, which may affect the activity of the enzyme.

3. For your safety and health, please wear a lab coat and disposable gloves when operating.

The product is for scientific research purposes only and not for clinical diagnosis!

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