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.

Inoculate 2-5 ml of L broth containing the appropriate antibiotic from a single bacterial colony. Incubate at 37℃ overnight with vigorous shaking.

Follow the Plasmid Quick Prep protocol through the potassium acetate step to isolate plasmid DNA.

Add DNAse-free RNAse to 50µg/ml, incubate, 37℃ from 10-30 minutes.

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℃, and rinse the pellets once with 70% ethanol. Respin the pellets for 1 min and dry under vacuum.

Resuspend pellets in 20µl H ₂ O 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℃, and Darise Farris reports good results at 85℃ for 5 min then cooling on ice.

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.

Centrifuge at room temperature or 4℃ 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.

Resuspend the pellet in 7 µl H ₂ O, 1 µl of pUC (or the appropriate) primer at a 0.5 pmol/µl stock concentration, and 2 µl of 5x Sequenase buffer. Mix and centrifuge to remove any debri. Heat 2 min, 65℃ and cool slowly to below 35℃ 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℃.

Follow the Sequenase protocol from this point. Darise Farris recommends the following modifications for optimizing for short sequences: dilute the labelling 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

RNAse (10 mg/ml)

Dissolve RNAse A in water. Boil for 5 minutes. Store at -20℃. 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 H₂ O. QS to 250 ml with water and autoclave.

3M Sodium Acetate, pH 5.5

18.46 g sodium acetate

Adjust pH with glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.