Description
Phi29 DNA polymerase, derived from Bacillus subtilis phage, has been genetically engineered to exhibit excellent strand displacement and continuous synthesis capabilities, enabling the synthesis of DNA fragments up to 70 kb. Additionally, it has strong 3'→5' exonuclease proofreading activity, thus it is recommended to modify the 3' end of primers in the system to prevent degradation. It is commonly used for in vitro plasmid synthesis and whole genome synthesis.
Specifications
| Catalog Numbers | 14404ES72 / 14404ES80 / 14404ES90 |
| Specifications | 250 U / 1,000 U / 5,000 U |
| Unit Definition | The amount of enzyme required to incorporate 0.5 pmol of dNTP into acid-insoluble precipitate in 10 minutes at 30°C. |
Components
| Component | Name |
14404ES72 250 U |
14404ES80 1000 U |
14404ES90 5000 U |
| 14404-A | Phi29 DNA Polymerase (10 U/μL) | 25 μL | 100 μL | 500 μL |
| 14404-B | 10×phi29 Reaction Buffer | 100μL | 400 μL | 2×1mL |
Heat Inactivation: 65°C for 10 minutes.
Storage
This product should be stored at -25~-15℃ for 2 years.
Instructions for Use
For genomic DNA amplification:
1. Reaction System Setup
Component Amount
10×phi29 Reaction Buffer 2 μL
Random Primers Total input 20-75 μM
dNTPs Total input 2 mM
Genomic DNA 5-50 ng
ddH2O to 19 μL
2. Place the reaction system in a PCR machine, incubate at 95°C for 5 minutes, and then quickly cool on ice for 2 minutes to denature the DNA template*.
3. Add 1-2 μL phi29 DNA Polymerase (10 U/μL) to the system and incubate at 30-42°C for 3 hours**.
4. Inactivate phi29 DNA polymerase by heating at 65°C for 10 minutes.
* Pre-denaturation of templates such as genomic and plasmid DNA is optional.
** The enzyme achieves maximum amplification yield at 42°C.
Precautions
1. It is normal for a small amount of precipitation to appear when the buffer is thawed. Please mix thoroughly before use.
2. This product is for research use only.
3. For your safety and health, please wear a lab coat and disposable gloves while handling this product.