Description
TaqIT DNA Polymerase is an exonuclease-deficient derivative of Taq DNA polymerase. The enzyme is supplied in 20 mM Tris-HCl, 222 mM (NH4)2SO4, 10 mM β-mercaptoethanol, 0.1 mM EDTA, 0.1% Brij 58 and 50% glycerol; pH 8.6 at 25°C. The 10x TaqIT Reaction Buffer contains 500 mM Tris-HCl, 160 mM (NH 4) 2 SO 4, 35 mM MgCl 2 and 0.25% Brij 58; pH 9.2 at 25⁰C.
Storage temperature: –25°C to –15°C Molecular weight: 62,407 Daltons Test Units tested Specification Purity n/a >99% Specific activity n/a 42,000 U/mg Single-stranded exonuclease 500 <1% released Double-stranded exonuclease 500 <1% released Double-stranded endonuclease 500 No conversion E. coli DNA contamination 500 <10 copies
TaqIT DNA Polymerase is an exonuclease-deficient derivative of Taq DNA polymerase. TaqIT DNA Polymerase lacks the first 280 amino acids of native Taq polymerase that contain the 5ʹ→3ʹ exonuclease domain. This deletion makes the TaqIT enzyme slightly more thermostable and has slightly greater fidelity than the full-length Taq enzyme. Like Taq polymerase, TaqIT DNA Polymerase has no inherent 3ʹ→5ʹ exonuclease activity. A recombinant E. coli strain carrying the TaqIT gene from the thermophilic organism Thermus aquaticus YT-1. One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C.
Usage instructions Set up the following reaction mixture in a total volume of 50 µL Component Volume (µL) Final concentration Nuclease-free H 2 O x N/A 10x TaqIT Reaction Buffer (B7620) 5 1x 10 mM dNTP mix 1 200 µM each Primer 1 x 0.2 µM Primer 2 x 0.2 µM DNA template x < 1000 ng TaqIT (P7620L) 0.1–0.5 5-25 U Note: Volumes can be adjusted as needed. Cycling conditions Step Temperature Time Cycles Initial Denaturation 94°C 30 s – 3 min 1 Denaturation 94°C 30 s 25–40 Annealing 50–65°C 30 s Extension 72°C 60 s/kb Final Extension 72°C 5 minutes hold 1 4°C Note: Cycling conditions may need to be optimized depending on the amplicon of interest. Quality Control Specific activity was measured using a two-fold serial dilution method. The enzyme was diluted in a reduced-glycerol (5%) containing TaqIT DNA Polymerase storage solution and added to 50 µL reactions containing calf thymus DNA, 25 mM TAPS (pH 9.3), 50 mM KCl, 1 mM DTT, 3H-dTTP and 100 µM dNTPs. Reactions were incubated for 10 minutes at 75°C, placed 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 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 band's mass 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.
