Preparation of plasmid DNA for sequencing.

S. Grayburn

Reference: Lee, S. and S. Rasheed. (1990) A simple procedure for maximum yield of high-quality plasmid DNA. BioTechniques 9:676-679.

Autoclave reagents, pipet tips, and centrifuge tubes before using.

(1) Inoculate 100 mL of Terrific Broth with E. coli from a fresh plate or glycerol stock. Medium should contain appropriate antibiotics to select for plasmid DNA. Grow bacteria overnight at 37 C, 200 rpm in a 500 mL flask. The next morning, cultures should appear turbid.
(2) Collect cells by filling two oak ridge tubes and centrifuge 10,000 rpm for 5 min at room temperature in a JA20 rotor. Decant supernatant and treat it as recombinant waste. Add the remainder of the 50 ml of cells per tube and recentrifuge. Decant the supernatant then remove the small amount of remaining supernatant with a pipettor.
(3) Resuspend each of the 2 pellets in 6.25 mL TEG (50 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA) with freshly added lysozyme at 5 mg/ml. Vortex until the pellet is broken up. Incubate at room temperature for 5 min.
(4) Add 12.5 mL 0.2 N NaOH, 1% SDS to the above solution , mix by inverting 5 times, and incubate on ice for 5 min.
(5) Add 9.375 mL ice cold 7.5 M ammonium acetate (pH 7.6) to the above solution, mix by inverting 5 times, and incubate on ice for 5 min.
(6) Centrifuge at room temperature for 10 min. at 10000 rpm.
(7) Transfer the clear supernatant to a new oak ridge tube. Fill the remainder of the tube with isopropanol, mix by inversion, and incubate at -80o for 10 min. or at -20 degrees indefinitely.
(8) Centrifuge at room temperature for 10 min. at 10000 rpm. Discard the supernatant and invert the tube on a clean paper towel to remove excess moisture.
(9) Add 3.125 ml of 2M ammonium acetate (pH 7.4) to the pellet and vortex extensively. Nucleic acids that are not solubilized at this point will be discarded with the pellet in the next step. Incubate on ice for 10 min. Vortex several times.
(10) Centrifuge at 4o for 10 min. at 10000 rpm. Save the supernatant to a new tube. (Autoclave the pellet).
11) Add 3.125 mL isopropanol, mix, and incubate at -80 C for 15 min.
12) Centrifuge at 4 C for 10 min. at 10000 rpm. Discard the supernatant and drain the tube onto a clean towel.
13) Add 0.4 mL 10 mM tris, 1 mM EDTA to each tube and dissolve the nucleic acids. Some nucleic acids may be on the side of the tube. Transfer the nucleic acids to a 1.5 mL tube.
14) Repeat step 13 with fresh buffer and a fresh 1.5 mL tube
15) Extract nucleic acids with 0.4 mL chloroform-vortex 30 sec., spin 5 min., and save top phases to new tubes.
16) Add 0.04 mL 3.14 M sodium acetate and 1 mL (95%) ethanol to each tube. Vortex. Chill at -80 C for 10 min then centrifuge at 4 C for 15 min.
17) Wash pellets with 70% ethanol and dry in speedvac.
18) Resuspend each pellet in 0.2 mL T.1E RNAse A and RNAse T1 and incubate at 37 C for 15 minutes after the pellets are dissolved. Combine identical nucleic acid samples (there should now be about 0.4 mL per tube).
19) Repeat steps 15-17.
20) Resuspend dry pellets in 0.1 mL 10 mM tris, 0.1 mM EDTA. Larger pellets may need up to 0.4 mL of buffer.
21) Measure DNA concentrations fluorometrically. A 1:10 dilution may be needed to measure the concentration of samples that have lots of DNA.