Manta 1.0 DNA Polymerase

• Thermostable DNA polymerase (Bst)
• No 5'→3' exonuclease activity
• No 3'→5' exonuclease activity

Manta 1.0 DNA Polymerase is a Thermostable bacillus (Bst) DNA polymerase that exhibits strong strand displacement. This thermophilic polymerase is deficient in both proofreading (3ʹ→ 5ʹ) and nick-translation (5ʹ→ 3ʹ) nuclease activities. The protein was originally characterized and its crystal structure solved by Lorena Beese (1).

The enzyme is supplied in 10 mM Tris-HCl, 50 mM KCl, 1.0 mM DTT, 0.1 mM EDTA, <0.1% Triton X-100 and 50% glycerol; pH 7.5 at 25°C.

10X PCR Buffer II (cat. no. B7140) contains 200 mM Tris-HCl, 100 mM (NH₄)₂SO₄, 100 mM KCl, 20 mM MgSO₄, and 1.0% Triton X-100; pH 8.8 at 25°C.

Ask about low glycerol formulations

Polymerase properties

• Storage temperature: –25°C to –15°C

Source of recombinant enzyme protein
The protein is produced by a recombinant E. coli strain carrying the Manta 1.0 DNA Polymerase (exo-) polymerase gene.

Unit definition:
One unit is defined as the amount of polymerase required to convert 10 nmol of dNTPs into acid insoluble material in 30 minutes at 65°C.

Molecular weight: 66,215 Daltons

Quality control analysis

Unit activity was measured using a twofold serial dilution method. Dilutions of enzyme batch were made in 1X reaction buffer and added to 50 µl reactions containing calf thymus DNA, 1X PCR Buffer II, 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 65°C, plased on ice and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26)

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.

• Strand displacement