EquiPhi29™ DNA Polymerase, 250 U - FAQs

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

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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.

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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.

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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.

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

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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

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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.

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

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

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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.

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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.

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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.

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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.

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

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