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?
How is phi29 Polymerase inactivated?
How is DNA denatured in phi29 DNA Polymerase reactions?
Can phi29 Polymerase be used to generate single-stranded DNA?
Can phi29 DNA Polymerase be used to amplify large, circular DNA constructs such as BACs and Fosmids?
Does the input DNA template have to be circular?
Can phi29 DNA Polymerase be used to blunt DNA?
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.