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 (OD260). If the primer has N bases, the approximate concentration (pmol/∝l) is given by the following formula:
Concentration (pmol/∝l)=OD260/(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 33P sequencing, a large tray for washing the gel (to remove urea) and a gel drying apparatus are highly recommended. For best results, gels containing 33P 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).