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Summary: 55
DNA Sequencing With Sequenase
Alkaline Denaturation of Plasmid dsDNA
1. Begin with 1-3 µg of plasmid DNA in 1-10 µl of TE. Optimal template is 0.5 pmol (e.g., 1 µg of 3
kb plasmid). Adjust to final volume of 30 µl with ddH2O. Set up in 0.5 ml tube.
2. Add 10 µl of 1 M NaOH. Vortex. Incubate at 25°C for 5 min. For plasmids > 5 kb, incubate at 37°C.
3. Add 60 µl of 4 M NH4OAc, pH 5. Vortex. Do not use pH 7 NH4OAc.
4. Add 250 µl of 100% EtOH. Vortex. Incubate at -70°C for 15 minutes (or more).
5. Spin at top speed for 30 minutes at 4°C. Remove supernatant carefully.
6. Add 150 µl of 70% EtOH. Vortex. Spin at top speed for 5 minutes at 4°C.
7. Dry pellet in Speed-Vac for 2-5 minutes. Cover tubes with parafilm with holes.
Annealing
1. Resuspend the DNA in 7 µl of ddH2O.
2. Add 2 µl of 5X Sequencing Reaction Buffer.
3. Add 1 µl (1.0-2.0 pmol) of primer. Vortex.
4. Heat to 65°C for 2 minutes, slowly cool to ~25°C over ~30 minutes. Spin briefly. Place on ice.
Can be stored at 0-4°C overnight.
Labeling Reaction
1. Combine on ice: 10 µl of Template DNA
1 µl of 0.1 M DTT
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