biotechrabbit™ phi29 DNA Polymerase is an exceptionally pure DNA polymerase for demanding applications. The enzyme is purified from a recombinant E. coli strain carrying the phi29 DNA Polymerase gene from bacteriophage phi29.
The enzyme is a highly processive DNA polymerase (up to 70,000 base insertions per binding event) with a powerful strand-displacement activity and a 3'→ 5' proofreading exonuclease function.
phi29 DNA Polymerase proofreading activity acts preferentially on single-stranded DNA or RNA. Therefore, to avoid primer degradation during the DNA synthesis, 3' modified (protected) primers are highly recommended.
Component | Composition |
phi29 DNA Polymerase | phi29 DNA Polymerase, 10 U/µl, in storage buffer containing 50% (v/v) glycerol. |
10× phi29 Reaction Buffer | Optimized 10× phi29 reaction buffer. |
STORAGE | -20°C (until expiry date – see product label) |
Unit Definition
One unit is defined as the amount of polymerase required to convert 0.5 pmol of dTTP into acid insoluble material in 10 minutes at 30°C.
Quality Control
Protein Purity
Protein purity is analyzed by SDS polyacrylamide gel electrophoresis.
Exonuclease Activity
Linearized lambda/HindII fragments are incubated with the enzyme in a 50 µl reaction mixture for 4 h at 37°C. No degradation of DNA was observed.
Endonuclease Activity
lambda DNA is incubated with the enzyme in a 50 µl reaction mixture for 4 h at 37°C. No degradation of DNA was observed.
Nick Activity
Supercoiled plasmid DNA is incubated with the enzyme in a 50 µl reaction mixture for 4 h at 37°C. No conversion of covalently closed circular DNA to nicked DNA was detected.
Contamination with E. coli DNA
A sample of denatured enzyme is analyzed with specific primers targeting the 16S rRNA gene in qPCR for the presence of contaminating E. coli DNA. No E. coli DNA was detectable.
References
- Blanco, L., et al. (1989) J. Biol. Chem., 264, 8935–8940.
- Garmendia, C., et al. (1992) J. Biol. Chem., 267, 2594–2599.
- Skerra, A., (1992) Nucleic Acids Res., 20, 3551–3554.
- Blanco, L., and Salas, M., (1984) Proc. Natl. Acad. Sci. USA, 81, 5325–5329.
- Lizardi, P.M., et al. (1998) Nat. Genet., 19, 225–232.
- Dean, F.B., et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 5261–5266.
- Simmel, F.C., et al. (2005) Nano Lett., 5, 719–722.
- Blanco, L., et al. (1994) Proc. Natl. Acad. Sci. USA, 91, 12198–12202.
- Lagunavicius, A., et al. (2009) RNA, 15, 765–771.
- Esteban, J.A., et al., (1993) J. Biol. Chem., 268, 2719 -2726.
Application recommendation
- Refer to the public articles for recommendations and protocols.
- Use of this enzyme in certain applications may be covered by patents and may require a license. Refer to the public articles for recommendations and protocols.
- The enzyme is heat inactivated by incubation at 65°C for 10 minutes.
1. Blanco, L. et al. (1989) J. Biol. Chem., 264, 8935-8940.
2. Garmendia, C. et al. (1992) J. Biol. Chem., 267, 2594-2599.
3. Skerra, A., (1992) Nucleic Acids Res., 20, 3551-3554.
4. Blanco, L. and Salas, M. (1984)Proc. Natl. Acad. Sci. USA, 81, 5325-5329.
5. Lizardi, P. M., et al. (1998) Nat. Genet., 19, 225-232.
6. Dean, F.B., et al., (2002) Proc. Natl. Acad. Sci. USA, 99, 5261-5266.
7. Simmel, F.C., et al., (2005) Nano Letters, 4, 719-722.
8. Blanco, L., et al., (1994) Proc. Natl. Acad. Sci. USA, 91, 12198-12202.
9. Lagunavicius, A., et al., (2009) RNA, 12, 765-771.
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