This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001
INTRODUCTION
Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains).
MATERIALS
Antibiotic for plasmid selection
Chloramphenicol (34 mg/ml)
Ethanol
Optional, please see Step 5.
Isopropanol
Lysozyme (10 mg/ml)
Prepare the solution fresh in Tris-Cl (pH 8.0).
Restriction endonucleases
Please see Step 4.
Rich medium
STE
STET
TE (pH 8.0)
METHOD
- 1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic either with a single colony of transformed bacteria or with 0.1-1.0 ml of a small-scale liquid culture grown from a single colony.
- 2. Incubate the culture at the appropriate temperature with vigorous shaking (250 cycles/ minute in a rotary shaker) until the bacteria reach the late log phase of growth (i.e., an OD600 of approx. 0.6).
- 3. Inoculate 500 ml of LB, YT, or Terrific Broth (prewarmed to 37°C) containing the appropriate antibiotic in a 2-liter flask with 25 ml of the late-log-phase culture. Incubate the culture for 2.5 hours at 37°C with vigorous shaking (250 cycles/minute on a rotary shaker).
- 4. Add 2.5 ml of 34 mg/ml chloramphenicol. The final concentration of chloramphenicol in the culture should be 170 µg/ml. Incubate the culture for a further 12-16 hours at 37°C with vigorous shaking (250 cycles/minute on a rotary shaker).
- 5. Remove an aliquot (1-2 ml) of the bacterial culture to a fresh microcentrifuge tube and store at 4°C. Harvest the remainder of the bacterial cells from the 500-ml culture by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. Discard the supernatant. Stand the open centrifuge bottle in an inverted position to allow all of the supernatant to drain away.
- 6. Resuspend the bacterial pellet in 200 ml of ice-cold STE. Collect the bacterial cells by centrifugation as described in Step 5. Store the pellet of bacteria in the centrifuge bottle at -20°C.
- 7. Prepare plasmid DNA from the 1-2-ml aliquot of bacteria set aside in Step 5 by the minipreparation protocol (either Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparationor Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation through NaCl). Analyze the minipreparation plasmid DNA by digestion with the appropriate restriction enzyme(s) to ensure that the correct plasmid has been propagated in the large-scale culture.
- 8. Allow the frozen bacterial cell pellet from Step 6 to thaw for 5-10 minutes at room temperature. Resuspend the pellet in 10 ml of ice-cold STET. Transfer the suspension to a 50-ml Erlenmeyer flask.
- 9. Add 1 ml of a freshly prepared solution of 10 mg/ml lysozyme.
- 10. Use a clamp to hold the Erlenmeyer flask over the open flame of a Bunsen burner until the liquid just starts to boil. Shake the flask constantly during the heating procedure.
- 11. Immediately immerse the bottom half of the flask in a large (2-liter) beaker of boiling water. Hold the flask in the boiling water for exactly 40 seconds.
- 12. Cool the flask in ice-cold water for 5 minutes.
- 13. Transfer the viscous contents of the flask to an ultracentrifuge tube (Beckman SW41 or its equivalent). Centrifuge the lysate at 150,000g (30,000 rpm in a Beckman SW41Ti rotor) for 30 minutes at 4°C.
- 14. Transfer as much of the supernatant as possible to a new tube. Discard the viscous liquid remaining in the centrifuge tube.
- 15. (Optional) If the supernatant contains visible strings of genomic chromatin or flocculent precipitate of proteins, filter it through 4-ply gauze before proceeding.
- 16. Measure the volume of the supernatant. Transfer the supernatant, together with 0.6 volume of isopropanol, to a fresh centrifuge tube(s). Store the tube(s) for 10 minutes at room temperature, after mixing the contents well.
- 17. Recover the precipitated nucleic acids by centrifugation at 12,000g (10,000 rpm in a Sorvall SS-34 rotor) for 15 minutes at room temperature.
Salt may precipitate if centrifugation is carried out at 4°C.
- 18. Decant the supernatant carefully, and invert the open tube(s) on a paper towel to allow the last drops of supernatant to drain away. Rinse the pellet and the walls of the tube(s) with 70% ethanol at room temperature. Drain off the ethanol, and use a Pasteur pipette attached to a vacuum line to remove any beads of liquid that adhere to the walls of the tube(s). Place the inverted, open tube(s) on a pad of paper towels for a few minutes at room temperature. The pellet should still be damp.
- 19. Dissolve the pellet of nucleic acid in 3 ml of TE (pH 8.0).
- 20. Purify the crude plasmid DNA either by chromatography on commercial resins (Purification of Plasmid DNA by Chromatography), precipitation with polyethylene glycol (Purification of Plasmid DNA by Precipitation with Polyethylene Glycol), or equilibrium centrifugation in CsCl-ethidium bromide gradients (Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradientsand Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients).
- 21. Check the structure of the plasmid by restriction enzyme digestion followed by gel electrophoresis.
REFERENCES
1. Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193–197.
| Caution |
Lysozyme
Lysozyme is caustic to mucous membranes. Wear appropriate gloves and safety glasses.
| Caution |
Radioactive substances
Radioactive substances: When planning an experiment that involves the use of radioactivity, consider the physico-chemical properties of the isotope (half-life, emission type, and energy), the chemical form of the radioactivity, its radioactive concentration (specific activity), total amount, and its chemical concentration. Order and use only as much as needed. Always wear appropriate gloves, lab coat, and safety goggles when handling radioactive material. X-rays and gamma rays are electromagnetic waves of very short wavelengths either generated by technical devices or emitted by radioactive materials. They might be emitted isotropically from the source or may be focused into a beam. Their potential dangers depend on the time period of exposure, the intensity experienced, and the wavelengths used. Be aware that appropriate shielding is usually made of lead or other similar material. The thickness of the shielding is determined by the energy(s) of the X-rays or gamma rays. Consult the local safety office for further guidance in the appropriate use and disposal of radioactive materials. Always monitor thoroughly after using radioisotopes. A convenient calculator to perform routine radioactivity calculations can be found at:http://www.graphpad.com/calculators/radcalc.cfm.
| Recipe |
Lysozyme
(10 mg/ml) Dissolve solid lysozyme at a concentration of 10 mg/ml in 10 mM Tris-Cl (pH 8.0) immediately before use. Make sure that the pH of the Tris solution is 8.0 before dissolving the protein. Lysozyme will not work efficiently if the pH of the solution is less than 8.0.
| Recipe |
Rich
LB
YT
SOB
For solid medium, please see Media Containing Agar or Agarose.
| Recipe |
STE
10 mM Tris-Cl (pH 8.0)
0.1 M NaCl
1 mM EDTA (pH 8.0)
Sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Store the sterile solution at 4°C.
| Recipe |
STET
0.1 M NaCl
5% (v/v) Triton X-100
10 mM Tris-Cl (pH 8.0)
1 mM EDTA (pH 8.0)
Make sure that the pH of STET is 8.0 after all ingredients are added. There is no need to sterilize STET before use.
| Recipe |
TE buffer, 10X
100 mM Tris-Cl (desired pH)
10 mM EDTA (pH 8.0)
Sterilize solutions by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Store the buffer at room temperature.
