DNA Sequencing Sequencing reaction

Tuesday, November 18, 2003

Description
Sequencing reaction

Procedure
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 65C 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 37C.

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 4C 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 37C for 2-5 minutes.

7) After termination, 5ul stop solution should be added to each tube, mixed by pipetting and the samples stored at -20C for later use.

Recipes
Freshly made 2M NaOH
3M sodium acetate, pH 4.5
Sterile, distilled water
Absolute ethanol
70% ethanol
7 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)

Supplies

Tips

(责任编辑：泉水)

搜索

热门标签:

DNA Sequencing Sequencing reaction