GE 33 Recording Equipment User Manual


 
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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/µl, 0.0006U/µl
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 MgCl
2
dGTP Nucleotide Master Mix: 7.5µM dATP, dCTP, dGTP, dTTP
dITP Nucleotide Master Mix: 7.5µM dATP, dCTP, dTTP, 37.5µM dITP
Stop Solution: 95% formamide, 20mM EDTA, 0.05% bromophenol blue, 0.05%
xylene cyanol FF
Control DNA: double-stranded pUC18, 0.02µg/µl
Control Primer (-40 M13 forward; 23-mer): 2.0pmol/µl
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 tolerant
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DNA sequencing gel. See
‘Supplementary Information, denaturing gel electrophoresis’ section.
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P-labeled Terminators: A package of four
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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, 0.3µM [α-
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P]ddGTP (1500Ci/mmol, 450µCi/ml), Redivue™
ddATP, 0.3µM [α-
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P]ddATP (1500Ci/mmol, 450µCi/ml), Redivue
ddTTP, 0.3µM [α-
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P]ddTTP (1500Ci/mmol, 450µCi/ml), Redivue
ddCTP, 0.3µM [α-
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P]ddCTP (1500Ci/mmol, 450µCi/ml), Redivue