Phi29 DNA Polymerase
This enzyme is a thermophilic DNA polymerase cloned from the Bacillus subtilis bacteriophage Phi29. It was expressed in Escherichia coli using gene recombination technology and purified and separated multiple times. Phi29 DNA polymerase has efficient DNA synthesis ability and can continuously synthesize up to 70kb of DNA fragments. At the same time, this enzyme has a 3 '→ 5' exonase reading function, high fidelity, and special chain displacement and continuous synthesis characteristics.
Product composition:
Component JN0020-125U
Phi29 DNA polymerase 125U
DNTP mixture (10mM each) 200 μ L
ten × Phi29 Buffer 1ml
Product features:
1. Extremely high incorporation rate.
2. Super strong chain permutation ability.
3. High fidelity.
4. React at medium temperature.
Product Usage:
1. Thermostatic protein primed DNA amplification.
2. Amplify DNA using random primers.
3. Roll ring replication.
4. Copy extended region.
Precautions:
1. The enzyme buffer contains a reducing agent DTT to ensure its maximum enzyme activity. Therefore, if the buffer is not fresh or has undergone repeated freeze-thaw cycles, 4MM of DTT should be added before use.
2. For reactions that require high amplification efficiency, please choose the upgraded version of Super phi29 DNA polymerase (JN0019) modified by Biotech Protein Engineering.
Application: Preparation of bacterial liquid sequencing template
Advantages: No need for bacterial cultivation, collection, lysis, and separation and purification of plasmid DNA, no need for centrifuges and PCR machines.
Sample pre denaturation: Select a monoclonal and refer to the thermal denaturation of the reaction system on the right.
Plasmid amplification: According to the following reaction conditions, the reaction time can be reduced to 1 hour if using Biolab super phi29 DNA polymerase (JN0019)*
Thermal deactivation: 65 ℃, 10 minutes.
Sequencing reaction: Take the above reaction solution 2 μ L plus 8 μ Double distilled water can be used for direct sequencing.
Common reaction systems (50 μ L)
ten × Phi29 Buffer 5 μ L
Random primer (1mM) 1.25 μ L
Plasmid template (1 μ G/ml) 1 μ L
Add ddH2O to 45 μ l. Pre denature at 95 ℃ for 3 minutes and cool on ice.
DNTP (10mM each) 2.5 μ L
100x BSA 0.5 μ L
Phi29 DNA Pol 2.5 μ L
Overnight at 30 ℃
Quality assurance: After multiple column purification, only clear and single target bands can be seen through SDS-PAGE gel detection; The PCR method detects no residual Escherichia coli DNA and no contamination of nucleic acid endonucleases or exonucleases.
Activity definition: The amount of enzyme required to blend 0.5 pmol of dNTP with acid insoluble substances within 10 minutes at 30 ℃ is defined as 1 active unit.
ten × Reaction buffer: 500mM Tris HCl (pH7.5), 100mM MgCl2
