COMPONENTS OF THE KIT

The solutions included in the Thermo Sequenase™ Radiolabeled Terminator Cycle Sequencing Kit have been carefully prepared to yield the best possible sequencing results. Each reagent has been tested extensively and its concentration adjusted to meet USB™ standards. It is strongly recommended that the reagents supplied in the kit be used as directed.

The following solutions are included in the kit:

Thermo Sequenase DNA Polymerase: 4U/∝l, 0.0006U/∝l Thermoplasma acidophilum inorganic pyrophosphatase**; 50mM Tris•HCl, pH 8.0, 1mM dithiothreitol (DTT), 0.1mM ethylenediamine tetraacetic acid (EDTA), 0.5% Tween™-20, 0.5% Nonidet™ P-40, 50% glycerol

Reaction Buffer (concentrate): 260mM Tris•HCl, pH 9.5, 65mM MgCl2

dGTP Nucleotide Master Mix: 7.5∝M dATP, dCTP, dGTP, dTTP

dITP Nucleotide Master Mix: 7.5∝M dATP, dCTP, dTTP, 37.5∝M dITP

Stop Solution: 95% formamide, 20mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF

Control DNA: double-stranded pUC18, 0.02∝g/∝l

Control Primer (-40 M13 forward; 23-mer): 2.0pmol/∝l 5'-GTTTTCCCAGTCACGACGTTGTA-3'

This kit and all the enclosed reagents should be stored at -15°C to -30°C (NOT in a frost-free freezer). Keep all reagents on ice when removed from storage for use. The kit can conveniently be stored at 2°C to 4°C for periods of up to 3 months with no loss of performance, but this should be avoided if it is expected that the reagents will not be completely consumed within 3 months.

Note: The formulation of Thermo Sequenase DNA polymerase in this kit necessitates the use of a glycerol tolerant8 DNA sequencing gel. See ‘Supplementary Information, denaturing gel electrophoresis’ section.

33P-labeled Terminators: A package of four 33 P-labeled terminators must be purchased for use with the kit. They may be ordered separately from GE Healthcare

using product number AH9539. In the US, the terminators

may be ordered together with the sequencing kit from USB using product number 188403.

ddGTP, 0.3∝M [α-33P]ddGTP (1500Ci/mmol, 450∝Ci/ml), Redivue™ ddATP, 0.3∝M [α-33P]ddATP (1500Ci/mmol, 450∝Ci/ml), Redivue ddTTP, 0.3∝M [α-33P]ddTTP (1500Ci/mmol, 450∝Ci/ml), Redivue ddCTP, 0.3∝M [α-33P]ddCTP (1500Ci/mmol, 450∝Ci/ml), Redivue

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