T4 DNA Polymerase

• OEM by QIAGEN offers bulk manufacturing of T4 DNA Polymerase in custom formulations
• 3′→5′ exonuclease activity
• No 5′→3′ exonuclease activity
• Ask about low-glycerol or glycerol-free formulations

T4 DNA Polymerase is the proven choice for polishing 5ʹ and 3 ʹ ends during DNA cloning. The enzyme catalyzes the extension of a primed DNA template in the 5ʹ→ 3ʹ direction. This enzyme exhibits a powerful 3ʹ→ 5ʹ exonuclease activity, while lacking any inherent 5ʹ→ 3ʹ exonuclease or strand displacement functions.

The enzyme is supplied in 100 mM KPO₄, 1.0 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 6.5 at 25°C.

10x Blue Buffer (cat. no. B0110): 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl₂ and 10 mM DTT; pH 7.9 at 25°C.

Polymerase properties

• Storage temperature: –25°C to –15°C

Source of recombinant enzyme protein
The protein is produced by a strain of E. coli that expresses the recombinant T4 DNA Polymerase gene.

Unit definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-precipitable material in 30 minutes at 37°C.

Molecular weight: 103,609 Daltons

References
1. Tabor, S., and Struhl, K. (1989). In Current Protocols in Molecular Biology Ausebel, F.M., et al (Eds.), pp. 3.5.10-3.5.12.
2. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 5.44-5.47.
3. Dale, R., McClure, B., and Houchins, J. (1985) Plasmid, 13:31.
4. Kunkel, T.A., Roberts, J.D., and Zakour, R.A., (1987) in Methods in Enzymology, R. Wu and L. Grossman (Eds.), 154, pp. 367-382. San Diego: Academic Press.
5. Panet, A., van de Sande, J.H., Loewen, P.C., and Khorana, H.G. (1973) Biochemistry, 12:5045.

Quality control analysis

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Very active 3′→5′ exonuclease activity
• Gap filing
• Creating blunt ends