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 fill in 3′ overhangs.

What is the extinction coefficient for phi29 DNA Polymerase?
The extinction coefficient for phi29 DNA Polymerase is 1.766 (mL/(mg*cm). If measuring protein concentration (A280), we recommend you use the protein’s extinction coefficient to ensure an accurate reading.
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 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.