TaqIT (5000 U)

From QIAGEN, this DNA polymerase is an exonuclease-deficient derivative of Taq DNA polymerase, suited for routine PCR amplification as well as reverse transcription from RNA for first-strand cDNA synthesis and RT-PCR. The enzyme lacks the first 280 amino acids of native Taq polymerase that contain the 5'→3' exonuclease domain, making it slightly more thermostable and conferring slightly greater fidelity than the full-length Taq enzyme. Like Taq polymerase, the enzyme has no inherent 3'→5' exonuclease activity, and is produced recombinantly from an E. coli strain carrying the TaqIT gene from Thermus aquaticus YT-1.

One unit is defined as the amount of enzyme that incorporates 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C. Quality control testing reports a specific activity of 42,000 U/mg and physical purity of >99% by SDS-PAGE and silver-stain detection. Contaminant assays report <1% released single-stranded exonuclease activity, <1% released double-stranded exonuclease activity, no conversion in a double-stranded endonuclease assay, and E. coli DNA contamination of <10 copies.

The enzyme is supplied in a buffer of 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), and is paired with a 10x Reaction Buffer containing 500 mM Tris-HCl, 160 mM (NH4)2SO4, 35 mM MgCl2 and 0.25% Brij 58 (pH 9.2 at 25°C). Recommended cycling conditions are an initial denaturation at 94°C for 30 s – 3 min (1 cycle), then 25–40 cycles of denaturation at 94°C for 30 s, annealing at 50–65°C for 30 s, and extension at 72°C for 60 s/kb, followed by a final extension at 72°C for 5 minutes.