phi29 Highlights
phi29 DNA Polymerase exhibits strong strand displacement activity and high processivity to enable efficient isothermal DNA amplification from low DNA template amounts. Its 3' → 5' exonuclease activity delivers high fidelity amplification, making it an appropriate solution for sequencing DNA template preparation.
- Strong strand displacement activity facilitates isothermal amplification
- Strong processivity delivers products up to 70 kb in length
- High-fidelity amplification supports DNA template preparation for sequencing
- Custom formats available, including high concentration
- Highly stringent enzyme production ensures quality performance across lots
Applications
- Multiple displacement amplification (MDA)
- Rolling circle amplification (RCA)
- Whole genome amplification (WGA)
- Cell-free cloning
- Preparation of DNA template for sequencing
Figure 1. Schematic of rolling circle amplification using phi29 DNA polymerase.
QC Specifications
| Description | Specification |
|---|---|
| Protein Purity Assay | ≥ 99% |
| Exonuclease Assay* | Functional |
| Specific Activity | 100,000 – 190,000 U/mg |
| DNA contamination Assay ( E. coli , mammalian, library)* | < 10 copies |
| Phosphatase Contamination Assay* | < 1% released |
| Endonuclease Contamination Assay* | Not detectable |
*As assessed using 1,000 U of enzyme input per assay.
Properties
Unit definition: One unit is defined as the amount of enzyme required to convert 50 pmol of dNTPs into a polynucleotide fraction in 10 minutes at 30°C
Reaction conditions:
1X phi29 Pol Reaction Buffer
Incubate at 30°C
Storage Buffer: 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 50% Glycerol, 0.5% Tween
1X phi29 Pol Reaction Buffer: 33 mM Tris-acetate (pH 8.2 at 25°C), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v/v) Tween 20, 1 mM DTT
Heat inactivation: 65°C for 10 min
Molecular weight: 66.3 kDa
5' – 3' Exonuclease activity: No
3' – 5' Exonuclease activity: Yes
Strand Displacement activity: High
Fidelity: High
Processivity: High
BROCHURES
phi29 DNA Polymerase Brochure
Isothermal Amplification Rolling Circle Amplification Single Cell Sequencing Enzymes phi29 DNAPWatchmaker Genomics' phi29 DNA Polymerase exhibits strong strand displacement activity and high processivity, enabling efficient isothermal DNA amplification from low template amounts. Its 3′→5′ exonuclease activity ensures high-fidelity amplification, making it suitable for sequencing DNA template preparation.
Precision Enzymes and Proteins Brochure
Custom Genomic Solutions Enzymes MDx DNA Polymerase I Equinox Library Amplification Equinox Uracil-Tolerant Library Amplification phi29 DNAP RNase Inhibitor StellarScript RT StellarScript HT RT StellarScript HT+ RT StellarTaq DNA Polymerase T4 DNA Ligase T4 DNAP T4 Polynucleotide Kinase T4 Gene 32 Protein Taq DNAPWatchmaker Genomics offers a portfolio of precision-engineered enzymes and proteins, designed to meet the demands of high-stringency applications. We provide tailored services such as custom fills, formats, packaging, and labeling to accommodate unique specifications.
PROTOCOLS
phi29 DNA Polymerase Technical Guide
Enzymes phi29 DNAPProtocol for Watchmaker Genomics' phi29 DNA Polymerase Kit which offers high-fidelity, isothermal DNA amplification with strong strand displacement and high processivity, ideal for applications like multiple displacement amplification (MDA), rolling circle amplification (RCA), whole genome amplification (WGA), cell-free cloning, and DNA template preparation for sequencing.
| CONFIGURATION | |
|---|---|
| Kit contents | phi29 DNA Polymerase (10 U/µL or 100 U/µL), 10X phi29 Pol Reaction Buffer (buffer not included in 100 U/µL kit) |
| Unit definition | One unit is defined as the amount of enzyme required to convert 50 pmol of dNTPs into a polynucleotide fraction in 10 minutes at 30°C. |
| Enzyme storage buffer | 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 50% Glycerol, 0.5% Tween |
| 10X phi29 Pol Reaction Buffer | 330 mM Tris-acetate (pH 8.2 at 25°C), 100 mM Mg-acetate, 660 mM K-acetate, 1% (v/v) Tween 20, 10 mM DTT |
| QUALITY CONTROL | |
|---|---|
| Protein Purity Assay | ≥ 99% |
| Exonuclease Assay | Functional |
| Specific Activity | 100,000 – 190,000 U/mg |
| DNA contamination Assay (E. coli, mammalian, library)* | <10 copies |
| Phosphatase Contamination Assay* | <1% released |
| Endonuclease Contamination Assay* | Not detectable |
*As assessed using 1,000 U of enzyme input per assay.
| SHIPPING AND HANDLING | |
|---|---|
| Shipping conditions | Ice packs |
| Storage | -20°C ± 5°C |
| Shelf life (off-the-shelf products) | 12 months |
| Shelf life (custom products) | ≥ 18 months |
What is phi29 DNA Polymerase?
Extremely processive B-type DNA Polymerase with DNA-dependent 5' → 3' DNA polymerase, strong strand displacement, and 3' → 5' exonuclease (proofreading) activity.
What is the unit definition of phi29 Polymerase?
One unit is defined as the amount of enzyme required to convert 50 pmol of dNTPs into a polynucleotide fraction in 10 minutes at 30°C.
What are the applications for phi29 DNA Polymerase?
- Multiple displacement amplification (MDA)
- Rolling circle amplification (RCA)
- Whole genome amplification (WGA)
- Cell-free cloning
- Preparation of DNA template for sequencing
What is the storage buffer for phi29 DNA Polymerase?
- 10 mM Tris-HCl, pH 7.4
- 0.1 mM EDTA
- 1 mM DTT
- 100 mM KCl
- 50% Glycerol
- 0.5% Tween
What is the recommended reaction buffer for phi29 DNA Polymerase?
- phi29 is supplied with a 10X reaction buffer:
- 330 mM Tris-acetate (pH 8.2 at 25°C)
- 100 mM Mg-acetate
- 660 mM K-acetate
- 1% (v/v) Tween 20
- 10 mM DTT
What are the recommended reaction setup conditions?
Please reference our technical guide for instructions for use.
| Component | Final Concentration | Volume (20 µL reaction) |
|---|---|---|
| DNA Template | Variable | Variable |
| Modified Primers | 2-5 µM | Variable |
| 10X phi29 Pol Reaction Buffer | 1X | 2 µL |
| dNTP Mix | 125 µM | 0.25 µL |
| phi29 DNA Polymerase (10 U/µL) | 0.5 U/µL | 1 µL |
| PCR-grade water | – | Up to 19 µL |
Are there recommendations for DTT usage?
DTT is required for activity of phi29 Polymerase. The reaction buffer contains DTT to ensure this activity. However, it is recommended that for buffer stocks older than 4 months or buffers that have undergone multiple freeze/thaw cycles DTT should be replenished by adding 10 μL 1M DTT per mL of reaction buffer.
What primers are recommended?
Protection of the 3′ ends of primers (hexamers are typically used) with at least two phosphorothioate internucleotide bonds is strongly recommended. DNA yield will significantly decrease if 3′-phosphorothioate modified primers are not used.
What primer concentration is recommended?
Optimal primer concentration depends on the application and thus should be optimized for each application.
For multiple displacement amplification (MDA), ~5 μM final primer concentration is recommended.
For rolling circle amplification (RCA), ~2 to 4 μM final primer concentration is recommended.
What is the optimal temperature for phi29 Polymerase?
The optimal temperature for phi29 Polymerase activity is 30°C. phi29 DNA Polymerase is thermolabile and we do not recommend reaction temperatures exceed 30°C.
How is phi29 Polymerase inactivated?
phi29 Polymerase is heat-inactivated at 65°C for 10 minutes.
How is DNA denatured in phi29 DNA Polymerase reactions?
DNA can be thermally denatured prior to addition of phi29 DNA Polymerase. An alternative to thermal DNA denaturation is alkaline DNA denaturation and neutralization. The 10X phi29 Pol Reaction Buffer is compatible with thermal and alkaline DNA denaturation.
Can phi29 Polymerase be used to generate single-stranded DNA?
Yes, by using unidirectional primers at a lower concentration of 0.2 to 1 μM final.
Can phi29 DNA Polymerase be used to amplify large, circular DNA constructs such as BACs and Fosmids?
Yes, phi29 can amplify larger, circular DNA constructs through rolling circle amplification (RCA). We recommend 2 to 4 μM final primer concentration for RCA.
Does the input DNA template have to be circular?
phi29 can amplify linear molecules through Multiple displacement amplification (MDA). We recommend altering the final primer concentration to 5 μM for MDA applications.
Can phi29 DNA Polymerase be used to blunt DNA?
Yes, phi29 produces blunt-end DNA fragments. However, DNA polymerase I and T4 DNA polymerase are the preferred enzymes for blunt-end protocol.
Can phi29 DNA Polymerase be used to fill in 3' overhangs?
No it cannot, 3' overhangs must be removed to create blunt ends. However, DNA polymerase I and T4 DNA polymerase can be used to remove 3' overhangs.
What is the extinction coefficient for phi29 DNA Polymerase?
Do you offer custom formats?
Yes! Watchmaker offers customer fills, packaging, concentrations, and labeling – including private label – designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our right-sized processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com to learn more about our capabilities.
Are you ISO 13485-certified?
Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.
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| Description | 0.25 kU | 1 kU | 5 kU | |
|---|---|---|---|---|
| phi29 DNA Polymerase Kit (10 U/μL)¹ incl. 10X phi29 Pol Reaction Buffer | 7K0104-25UL | 7K0104-100UL | Request a quote | |
| phi29 DNA Polymerase Kit (100 U/μL)¹ | 3K0106-50UL | Request a quote |
¹ A unit is defined as the amount of enzyme required to convert 50 pmol of dNTPs into a polynucleotide fraction in 10 minutes at 30°C.
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
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