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DNA Sequencing With Sequenase Alkaline Denaturation of Plasmid dsDNA
 

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

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine