DNA Sequencing

The sequencing reactions described below work perfectly well if you are short of cash to buy sequencing kits. It is based on the Dideoxy sequencing method of Sanger et al., 1977. However, due to the number solutions that need to be made, I recommend purch asing a sequencing kit, we use either the T7 Sequencing Kit (Pharmacia, 100 reactions) or the Sequenase 2.0 Sequencing Kit (USB via Amersham in the U.K., 100 reactions). Reactions are performed in sterile 1.5ml microcentrifuge tubes. Primers are synthesised on an Applied Biosystems 381A DNA synthesiser.
Approximately 3ug denatured, high quality dsDNA (i.e. prepared as described in 'Plasmid Isolation using PEG') are typically used for standard sequencing reactions.
DNA Sequencing Reactions
You will need:
Freshly made 2M NaOH3M sodium acetate, pH 4.5Sterile, distilled waterAbsolute ethanol70% ethanol7 x DNA annealing buffer (280mM Tris.Cl, pH 7.5, 100mM MgCl2, 350mM NaCl)Termination mixes (40mM Tris.Cl, pH 7.5, 50mM NaCl, 10mM MgCl2, 150mM dTTP, 150mM dATP, 150mM dCTP, 150mM c7deaza-dGTP and 15mM of the respective ddNTP)5 x DNA labelling mix (10mM dGTP, 10mM dCTP, 10mM dTTP, 200mM Tris.Cl, pH 7.5, 250mM NaCl)300mM DTT[a-35S]-dATP (~ 1000Ci/mmol, Amersham or DuPont)T7 DNA polymerase (Pharmacia)Stop solution (95% deionized formamide, 20mM EDTA, pH 7.5, 0.1% each of bromophenol blue and xylene cyanol FF)
1) Denature dsDNA by the addition of 8ul DNA (approximately 3ug) to a sterile microcentrifuge tube containing 2ul freshly made 2M NaOH vortexed briefly and incubate at room temperature for 10 minutes.
2) Neutralise DNA by the addition of 3ul 3M sodium acetate, pH 4.5 and 7ul sterile, distilled H2O and precipitate by the addition of 60ul ethanol. Recover DNA by centrifugation, at maximum speed, for 10 minutes in a microfuge. Rinse DNA briefly in 70% ethanol, air dry and re-dissolve in 10ul sterile, distilled H2O.
3) To a microcentrifuge tube containing 10ml denatured template DNA, add 4.44ng primer (2ul of a 2.22ng/ul stock) and 2ml 7 x annealing buffer. Heat the mixture to 65°C for 2 minutes and allow to cool slowly, over a period of about 30 minutes, to roo m temperature.
4) While the annealing reaction is taking place, take 4 sterile microcentrifuge tubes per sample and label G, A, T, C, respectively. Place into each tube 2.5ul, respectively, of the corresponding termination mix. Pre-warm tubes to 37°C.
5) After completion of the annealing reaction, the labelling reaction is initiated by the addition to the annealed template/primer of 2ul 1 x labelling mix, 1ul 300mM DTT, 1ul [a-35S]-dATP (~ 1000Ci/mmol) and 3 units T7 DNA polymerase (2ul of a 1.5 units/ ul solution). The solution is pipetted briefly to mix the components and incubated at 4°C for 2-5 minutes.
6) Termination is achieved by transferring 4.5ul of the labelling reaction into each of the 4 tubes labelled G, A, T, C, respectively and incubating at 37°C for 2-5 minutes.
7) After termination, 5ul stop solution should be added to each tube, mixed by pipetting and the samples stored at -20°C for later use.