Phi29 DNA polymerase
Phi29 DNA polymerase
Cat. No.: SB-G3448-300U
Size: 300U
Description
This product, phi29 DNA polymerase, is phi29 DNA polymerase, derived from Bacillus stearothermophilus bacteriophage phi29, recombinantly expressed in Escherichia coli, which has DNA strand replacement activity and strong DNA continuous synthesis ability (can realize continuous DNA with a length of more than 70 kb) Polymerization extension), with 3'-5' exonuclease activity, has strong fidelity, and is mainly used for rolling circle isothermal amplification (RCA), genome amplification sequencing, etc.
- Source : DNA Polymerase gene derived from Bacillus subtilis phage phi29, recombinantly expressed and purified by Escherichia coli.
- Definition of enzyme activity: at 30°C, the amount of enzyme required to incorporate 0.5 pmol dNTP into the acid-insoluble precipitate within 10 minutes is defined as one enzyme activity unit.
- Purity and concentration: SDS-PAGE detection purity ≥ 95%; endogenous nucleic acid residue < 10 -1 pg/μL (qPCR detection); 10 U/μL.
- Inactivation or inhibition: Heat at 65°C for 10 minutes 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. Effects of phi29 DNA Polymerase catalyzed plasmid (A) and 293 cell genome (B) DNA amplification. The reaction system is 20 μL, the template in A is the pUC19 plasmid, and the template in B is the 293 cell genome, and random primers are used for the primers. Incubate the whole system at 95°C for 5 min, and add 1 μL of phi29 DNA Polymerase (8, 4, 2, 1 times) with different dilution ratios after 2 min in ice bath, corresponding to numbers 3, 4, 5, and 6 respectively, and not added to number 1 phi29 DNA Polymerase, 1 μL phi29 DNA Polymerase was added to No. 2, but no template was added, and the system was incubated at 30°C for 2 h.
Storage and transportation
Transport in wet ice packs; store at -20°C, valid for 12 months.
Product Contents
| Component Number | Component | G3448-300U |
| G3448-1 | phi29 DNA Polymerase | 30 μL |
| G3448-2 | 10× phi29 Reaction buffer | 500 μL |
| Product Manual | 1 copy | |
Steps
1. Refer to the following table to prepare the reaction system:
| Component | Volume |
| Template DNA (>1 ng) | 1 μL |
| Random Primers (100 μM) | 1 μL |
| dNTP Mix (2.5 mM each) | 1 μL |
| 10×phi29 Reaction Buffer | 2 μL |
| Nuclease Free Water | To 20 μL |
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. (In general, 2 hours of incubation is enough, if you want to obtain a larger amount of product, you can extend the time to 16 hours)
4. End the reaction: Incubate at 65°C for 10 minutes.
5. Amplified products can be detected by agarose gel electrophoresis.
Precautions
2. The operation of enzyme products should be carried out in an ice bath to avoid affecting the activity of the enzyme when the enzyme is left at room temperature for a long time.
3. For your safety and health, please wear a lab coat and disposable gloves for operation.
