USB™ CycleSeq™ Thermostable DNA Polymerase (Applied Biosystems™)

USB™ CycleSeq™Thermostable DNA Polymerase is a thermostable, DNA polymerase that is exonuclease-free and incorporates ddNTPs with a marked level of efficiency over standard thermostable DNA polymerases. This allows for more even and easy-to-read sequence band patterns, making sequence anomalies easier to identify.

USB CycleSeq Thermostable DNA Polymerase can be substituted for ThermoSequenase DNA polymerase as both enzymes incorporate modified nucleotides (see Fig. 1).

CycleSeq DNA Polymerase, like Thermo Sequenase DNA Polymerase, includes Thermoplasma acidophilum inorganic pyrophosphatase (TAP). TAP is thermostable and converts pyrophosphate to orthophosphate, a useful feature during cycle sequencing reactions where pyrophosphates are produced.

USB CycleSeq Thermostable DNA Polymerase is useful for cycle sequencing (Sanger sequencing), as it produces sequence data with uniform band intensities which allows for longer and more accurate sequence reads. It is also useful for primer extension protocols during SNP genotyping.

Source:
Recombinant

Purity:
Greater than 95% pure as determined by SDS-PAGE.

Storage Buffer:
20 mM Tris-HCl, pH 8.5, 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.053 unit/μl Thermoplasma acidophilum inorganic pyrophosphatase, 50% glycerol, and stabilizers.

Assay Conditions:
The reaction mixture contains 25 mM TAPS, pH 9.3, 50 mM KCl, 2 mM MgCl₂, 1 mM Β-ME, 200 μM dATP, 200 μM dGTP, 200 μM dTTP, 100 μM dCTP, 0.05 μL [α33P] dCTP (10 μCi/μL), 400 μg/mL activated DNA, and CycleSeq Thermostable DNA Polymerase. After incubation at 74deg;C for 10 minutes, acid-insoluble material is determined (50 μL reaction volume).

Unit Definition:
One unit of enzyme is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol of dNTPs into acid-insoluble form in 30 minutes at 74°C under standard assay conditions.

Concentration:
32 units/μL

Functional test:
Functionally tested to meet or exceed the specifications for use in dye primer sequencing. Release specifications are based on sequence length, band intensity, and sequence quality. The sequence must also be free of background bands strong enough to interfere with sequence interpretation.