Biology Reference
In-Depth Information
Table 7.1: An Example of a Dideoxy (Sanger) Sequencing Protocol Using Modified
Bacteriophage T7 DNA Polymerase (Sequenase™).
Sequencing reagents
Annealing buffer (5 × concentrate)
  200 mM Tris•HCl (pH 7.5)
100 mM MgCl 2
250 mM NaCl
Dithiothreitol (DDT), 0.1 M
Labeling nucleotide mixture (for use with radiolabeled dATP)
1.5 mM dGTP, 1.5 mM dCTP, and 1.5 mM dTTP
Termination nucleotide mixtures (one for each dideoxynucleotide)
Each mixture contains 80 mM dGTP, 80 mM dATP, 80 mM dTTP, 80 mM dCTP, and 50 mM NaCl.
In addition, the “G” mixture contains 8 mM dideoxy-dGTP; the “A” mix, 8 mM ddATP; the “T,” 8 mM
ddTTP; and the “C,” 8 mM ddCTP.
Stop solution : 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol FF
Procedures
Annealing template and primer
1. For each template, a single annealing (and subsequent labeling) reaction is used. Combine the following:
Primer
0.5 pmol
DNA
0.5-1.0 pmol (1 mg of M13)
Annealing buffer
2 ml
Water to bring the total volume to 10 ml
Warm the capped tube to 65°C for 2 minutes, allow the mixture to cool slowly to room temperature over a
period of 30 minutes.
Labeling reaction
2. To the annealed template-primer add the following:
DTT (0.1 M) 1 ml
Labeling nucleotide mix 2 ml
α - 35 S or α - 32 P dATP 5 mCI (typically 0.5 ml)
Sequenase™ 3 units
Total volume should be 15 ml; mix thoroughly and incubate for 2-5 minutes at room temperature.
Termination reactions
3. Label four tubes “G,” “A,” “T,” and “C.” Fill each with 2.5 ml of the appropriate dideoxy termination
mixture.
When the labeling reaction is complete, transfer 3.5 ml of it to the tube prewarmed to 37°C, labeled “G.” Do
the same for each of the other three tubes (A, T, and C).
After 2-5 minutes at 37°C, add 4 ml of Stop Solution to each termination reaction, mix, and store on ice. To
load the gel, heat the samples to 75-80°C for 2 minutes or more and load 2-3 ml in each lane.
4. Polyacrylamide gel electrophoresis
5. Analysis of sequences
(Modified from Tabor and Richardson 1987.)
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