Thermo Scientific™ EquiPhi29™ DNA Polymerase

Catalog No.	Quantity
A39390	250 U
A39391	1000 U
A39392	5000 U

Catalog No. A39390 Supplier Thermo Scientific™ Supplier No. A39390

Description

Thermo Scientific EquiPhi29 DNA Polymerase is a proprietary phi29 DNA polymerase mutant developed through in vitro protein evolution.

Thermo Scientific EquiPhi29 DNA Polymerase is a proprietary phi29 DNA polymerase mutant developed through in vitro protein evolution. This enzyme is significantly improved over phi29 DNA polymerase in protein thermostability, reaction speed, product yield, and amplification bias, while retaining all the benefits of the wild-type enzyme.

EquiPhi29 DNA Polymerase possesses strong strand displacement activity and 3'→5' proofreading exonuclease activity that acts preferentially on single-stranded DNA or RNA.

Features of EquiPhi29 DNA Polymerase
• Highest processivity and strand displacement activity—more than 70 kb long DNA stretches can be synthesized
• Low amplification bias
• Extremely high yields of amplified DNA, even from small amounts of template
• Highly accurate DNA synthesis in a short time 

Applications
EquiPhi29 DNA Polymerase may be used in a wide variety of applications including:
• Unbiased whole genome amplification (WGA)
• Rolling circle amplification (RCA)
• Protein-primed DNA amplification
• Cell-free cloning of lethal DNA
• In situ genotyping with padlock probes
• RNA-primed DNA amplification

Notes
1. The addition of pyrophosphatase to the reaction mixture with EquiPhi29 DNA Polymerase may further enhance DNA synthesis
2. Because of the enzyme's 3'→5' proofreading exonuclease, 3’-modified primers are highly recommended.
3. EquiPhi29 DNA Polymerase can amplify DNA in a wide range of temperatures (30–45°C), with an optimal reaction temperature of 42°C.

Specifications

• Product Type: Stand-alone enzyme
• For Use With (Application): MDA-WGA, RCA
• Polymerase: EquiPhi29 DNA Polymerase
• Quantity: 250 U
• Content And Storage:

  • EquiPhi29 DNA Polymerase (25 μL at 10 U/μL)
  • 10X EquiPhi29 DNA Polymerase Reaction Buffer (0.25 mL)
  • 110 mM DTT (0.25 mL)
  
  Store at –15 to –25°C.
  
  

• Optimal Reaction Temperature: 42°C
• Shipping Condition: Dry Ice
• Concentration: 10 U/μL
• Reaction Time: 2 hours

Frequently Asked Questions (FAQs)

What is the error rate of EquiPhi29 DNA Polymerase?

The error rate of EquiPhi29 DNA Polymerase is 6 x 10^-6.
The error rate of EquiPhi29 DNA Polymerase was measured according to the method described in literature:
Mielinis, P., Sukackaitė, R., Serapinaitė, A., Samoilovas, F., Alzbutas, G., Matjošaitis, K., & Lubys, A. (2021). MUA-based molecular indexing for rare mutation detection by Next-Generation sequencing. Journal of Molecular Biology, 433(19), 167209. https://doi.org/10.1016/j.jmb.2021.167209

Can I use EquiPhi29 DNA Polymerase to incorporate 5-methyl-dCTP?

Thermo Scientific EquiPhi29 DNA Polymerase is a proprietary mutant phi29 DNA Polymerase developed through in vitro protein evolution. EquiPhi29 DNA Polymerase as well as phi29 DNA Polymerase should be able to incorporate 5-methyl-dCTP nucleotides and other modified nucleotides.

When using EquiPhi29 DNA Polymerase, do I need to purify amplified DNA products before downstream applications?

Cleaning of the amplified product is not required prior to several downstream methods (e.g., debranching, digestion with restriction endonucleases, Sanger sequencing); the dilution of amplified product is sufficient. If the clean-up procedure is needed, we recommend using an affinity-based spin-column or magnetic bead-based purification method.

What is the minimal recommended time for amplification with EquiPhi29 DNA Polymerase?

The optimal reaction time for DNA amplification with EquiPhi29 DNA Polymerase is 2 hours. For samples with ≥1 pg of DNA input, DNA amplification time can be shortened to 1 hour if maximizing product yield is not essential.

Can liquid media culture or colonies be used as a starting material for amplification with EquiPhi29 DNA Polymerase?

Yes. EquiPhi29 DNA Polymerase can work with different types of sample input material such as purified DNA, liquid media culture, agar plate colonies, etc.

Is EquiPhi29 DNA Polymerase suitable for single cell DNA amplification?

EquiPhi29 DNA Polymerase is suitable for amplification from low amounts of starting DNA material, making it ideal for single-cell analysis. Amplification from single cells using EquiPhi29 DNA polymerase can be reviewed in the literature:
Stepanauskas, R., Fergusson, E. A., Brown, J., Poulton, N., Tupper, B., Labonté, J. M., Becraft, E. D., Brown, J., Pachiadaki, M., Povilaitis, T., Thompson, B., Mascena, C. J., Bellows, W. K., & Lubys, A. (2017). Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles. Nature Communications, 8(1). https://doi.org/10.1038/s41467-017-00128-z

Is EquiPhi29 DNA Polymerase supplied with DTT?

Yes, 100 mM DTT is supplied as a separate component. The enzyme requires active reducing reagent in the reaction mix, therefore fresh DTT should be added separately. As DTT degrades over time, older DTT or DTT that has been frozen and thawed more than 10 times should be substituted with freshly prepared DTT.

Is EquiPhi29 DNA Polymerase supplied with dNTPs?

No. dNTPs can be purchased separately (Cat. No. R0181).

Can EquiPhi29 DNA Polymerase be heat inactivated?

Yes, we recommend heat inactivating EquiPhi29 DNA Polymerase at 65 degrees C for 10 min.

Can EquiPhi29 DNA Polymerase reaction volume be scaled up or scaled down?

Yes, the reaction volume can be scaled down to 10 µL and scaled up to 50 µL from the recommended 20 µL. Scaling up the reaction volume more than 4-fold may affect performance.

What is the amplification reaction temperature range for EquiPhi29 DNA Polymerase?

EquiPhi29 DNA Polymerase is a thermostable enzyme. The optimal reaction temperature range is 42-45 degrees C, however it will also amplify DNA at 30 degrees C.

How can I avoid non-specific DNA amplification with EquiPhi29 DNA Polymerase?

EquiPhi29 DNA Polymerase is a thermostable enzyme with a fast reaction speed. Amplification at 45 degrees C for 1-2 hr is sufficient to obtain specific product amplification without background amplification. The reaction is saturated after 3 hr of incubation.

Is phi29/EquiPhi29 DNA Polymerase suitable for amplification of short DNA fragments?

The amplification efficiency of MDA (multiple displacement amplification) reaction rapidly diminishes as the molecular weight of the starting material decreases, thus making the polymerase unsuitable for amplification of low-molecular weight DNA. However, it is suitable for amplification from extremely low amounts of starting DNA material, making it ideal for single cell analysis.

What type of DNA template can be amplified by EquiPhi29 DNA Polymerase?

Both linear and circular DNA templates can be amplified by EquiPhi29 DNA Polymerase.

For Research Use Only. Not for use in diagnostic procedures.