Mako DNA Polymerase

• OEM by QIAGEN offers bulk manufacturing of Mako DNA Polymerase in custom formulations
• QIAGEN’s version of T4 DNA Polymerase (exo –)
• Extends DNA in the 5′→3′ direction
• Lacks 3′→5′ and 5′→3′ exonuclease activities
• No strand displacement function

Mako DNA Polymerase is exonuclease deficient and lacks strand displacement activity. The enzyme catalyzes the extension of a primed DNA template in the 5ʹ→3ʹ direction. Inherent 3ʹ→5ʹ and 5ʹ→3ʹ exonuclease activities are absent and the enzyme exhibits no measurable strand displacement function.

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 (B0110) contains the following: 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl₂ and 10 mM DTT; pH 7.9 at 25°C.

Properties

• Storage temperature: –25°C to –15°C   
• Molecular weight: 103,565 Daltons

Source of recombinant enzyme protein
The protein is produced by a strain of E. coli that expresses the recombinant Mako DNA Polymerase (3ʹ→ 5ʹ exo–) 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.

Quality control analysis

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

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.

• Labeling
• Partial fill-in reactions