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MATERIALS NOT SUPPLIED
Necessary reagents:
Water—Only deionized, distilled water should be used for the sequencing
reactions.
Specialized sequencing primers—Some sequencing projects will require the
use of primers which are specific to the project. For most sequencing
applications, 0.5-2.5pmol of primer should be used for each set of sequencing
reactions. Always determine the concentration of the primer by reading the
optical density at 260nm (OD
260
). If the primer has N bases, the approximate
concentration (pmol/µl) is given by the following formula:
Concentration (pmol/µl)=OD
260
/(0.01 x N) where N is the number of bases.
Gel reagents—Sequencing gels should be made from fresh solutions of
acrylamide and bis-acrylamide. Other reagents should be electrophoresis grade
materials. For convenience, RapidGel™ gel mixes are strongly recommended.
RapidGel-XL formulations yield up to 40% more readable sequence per gel.
See ‘Related Products’ section for range of USB Ultrapure gel products.
Necessary equipment:
Liquid handling supplies such as vials, pipettes and a microcentrifuge—All
sequencing reactions are run in plastic microcentrifuge tubes (typically 0.5ml)
suitable for thermal cycling.
Electrophoresis equipment—While standard, non-gradient sequencing gel
apparatus is sufficient for much sequencing work, the use of field-gradient
(‘wedge’) or salt-gradient gels will allow much greater reading capacity on the
gel (4,5,17). A power supply offering constant voltage operation at 2000V or
greater is essential.
Gel handling—For
33
P sequencing, a large tray for washing the gel (to remove
urea) and a gel drying apparatus are highly recommended. For best results,
gels containing
33
P must be exposed dry in direct contract with the film at room
temperature.
Autoradiography—Any large format autoradiography film such as the
BioMax™ MR, and a large film cassette.
Thermal cycler—Sequencing will require thermally cycled incubations between
50°C and 95°C (1-100 cycles).