PREPARATION OF PLASMID DNA FOR SEQUENCING

The following protocol is based on our modifications of R. Kraft, J. Tardiff, K. S. Krauter, and L. A. Leinwand. Biotechn. 6(6):544-545, 1988.
  1. Inoculate 2-5 ml of L broth containing the appropriate antibiotic from a single bacterial colony. Incubate at 37°C overnight with vigorous shaking.
  2. Follow the Plasmid Quick Prep protocol through the potassium acetate step to isolate plasmid DNA.
  3. Add DNAse-free RNAse to 50µg/ml, incubate, 37°C from 10- 30 minutes.
  4. Phenol extract the solution, saving the upper aqueous phase. Ether extract the remaining phenol, and ethanol precipitate the DNA. Resuspend the pellets in 16.8 µl H2O, 3.2 µl 5 M NaCl, 20µl 13% PEG 8000, and incubate on ice for 30 min. Spin in a microfuge for 10 min, 4°C, and rinse the pellets once with 70% ethanol. Respin the pellets for 1 min and dry under vacuum.
  5. Resuspe nd pellets in 20µl H2O and 2 µl (1/10 volume) of a solution of 2 N NaOH and 2 mM EDTA. The original protocol called for a 5 min room temperature incubation, others incubate 30 min @ 37°C, and Darise Farris reports good results at 85°C for 5 min then cooling on ice.
  6. Neutralize with 1/10 volume of 3 M sodium acetate (pH 4.5-5.5) [or add 8 µl of 1 M Tris-HCl (pH 4.0-4.5), 3 µl of 3 M sodium acetate (pH 5.2)], and 75 µl of cold ethanol. Incubate on dry ice for 20 min.
  7. Centrifuge at room temperature or 4°C in a microfuge for 5 min. Discard the supernatant and wash the pellet with 70% ethanol and centrifuge 2 min. Discard the supernatant and dry the pellet under vacuum.
  8. Resuspend the pellet in 7 µl H2O, 1 µl of pUC (or the appropriate) primer at a 0.5 pmol/µl stock concentra tion, and 2 µl of 5x Sequenase buffer. Mix and centrifuge to remove any debri. Heat 2 min, 65°C and cool slowly to below 35°C by removing the heat block with samples in it from the heater to the lab bench. This cooling typically takes 30-45 min. Thaw the isotope during this time. Place tubes on ice when they reach 35°C.
  9. Follow the Sequenase protocol from this point. Darise Farris recommends the following modifications for optimizing for short sequences: dilute the labell ing mix 1:20 instead of 1:5, add 1 µl of Mn buffer (0.15 M sodium isocitrate & 0.1 M MnCl2) at the annealing step, and incubate the stop reactions for 15 min instead of 5 min.
RECIPES
L Broth (LB; Luria-Bertani)
10 g tryptone
5 g yeast extract
5 g NaCl
1 L water
Autoclave
RNA se (10 mg/ml)
Dissolve RNAse A in water. Boil for 5 minutes. Store at -20° C. Some protocols also add an equal volume of 40% glycerol and a half volume of 0.5 M NaCl.
5 M Sodium Chloride
73.06 g NaCl
Add NaCl gradually to 200 ml H2O. QS to 250 ml with water and autoclave.
3M Sodium Acetate, pH 5.5
18.46 g sodium acetate
Adjust pH w ith glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.
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Darise Farris, Ph.D. is gratefully acknowledged for her comments to this protocol.
Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Preparation of Plasmid DNA for Sequencing. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/dsdnaseq.html). 1997. Oklahoma City. Revision Date: October 2, 1997."