DNA Preparation, Plasmid DNA - Boiling Method
Jeff Lawrence
1. Grow 3 mL culture overnight in appropriate antibiotic. Pellet cells at 12 K for 10 min in table top centrifuge. Remove supernatant by aspiration. Resuspend pellet in 400 µL STET:
8 % Sucrose (40 mL 20% Sucrose)
10 mM Tris, pH 8.0 (1 mL 1 M Tris, pH 8.0)
50 mM EDTA (10 mL 500 mM EDTA, pH 8.0)
0.5 % Triton X-100 (5 mL 10% Triton X-100)
(44 mL ddH2O)
2. Add 20 µL of 20 mg/mL Lysozyme. Incubate at room temperature for 1 min.
3. Add 2 µL fresh DEPC. Vortex 30 sec.
4. Boil immediately for 40 sec. Chill quickly on ice.
5. Microfuge for 15 min at 4°. Remove pellet with a sterile toothpick or transfer supernatant to a new tube.
7. Add 1 mL 95 % Ethanol. Precipitate for 30 min at -20°. Microfuge for 15 min at 4°.
8. Discard supernatant. Dry in vacuum desiccator for 4 min. Resuspend pellet in 50 µL TE. DNA prepared by this method may not be clean enough for restriction digests.
Last Update: Thursday June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu