Description
Manta 1.0 DNA Polymerase is a Thermostable bacillus (Bst) DNA polymerase that exhibits strong strand displacement. This 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). 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. Supplied with: 10X PCR Buffer II (cat. no. B7140) contains 200 mM Tris-HCl, 100 mM (NH 4) 2SO 4, 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 Test Units Tested Specification Purity n/a >99% Specific activity n/a 400,000 U/mg Single-stranded exonuclease 4000 U <5.0% released Double-stranded exonuclease 4000 U <1.0% released Double-stranded endonuclease 4000 U no conversion E. coli DNA contamination 4000 U <10 copies
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
Usage Instructions: Manta 1.0 DNA Polymerase exhibits strong strand displacement activity and can be used in various nucleic acid amplification methods, such as isothermal, whole genome, and multiple displacement amplifications. Reaction Conditions Optimal reaction temperature is between 60-65°C. 10X Reaction Buffer contains 2 mM MgSO 4 at 1X. No dNTPs are included in the buffer. For optimization, add MgSO 4 up to 10 mM, and Manta 1.0 Polymerase up to 1.6 U/µL. Quality control analysis Unit activity was measured using a twofold serial dilution method. Dilutions of the 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. The reactions were incubated for 10 minutes at 65°C, placed on ice and analyzed using the method of Sambrook and Russell (2). Protein concentration is determined by OD 280 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 16S rDNA contamination i s 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.
