B31 Large Scale Plasmid Isolation

Purpose

To isolate plasmids on a large scale, from 100 mL to 1 L cultures.

Prior Work

Usually some transformed bacteria (A-22) with properties meriting extensive further study will be grown in large amounts prior to a large scale plasmid isolation.

Materials

Background

Procedure

IMPORTANT: USE PRECAUTIONS APPROPRIATE FOR THE BACTERIA YOU ARE WORKING WITH.

  1. Remove the culture of transformed bacteria from the shaker-incubator and transfer to a centrifuge bottle. Cap the tube. Prepare a balancing tube for centrifugation. Centrifuge at 4700 g for 10 minutes at 4°C. Centrifugation leads to the formation of a pellet of bacteria. Decant the supernatant fluid into the original culture flask.
  2. Add STE solution to wash the cells. Cap the tube. Resuspend the bacteria using a vortex mixer. Continue vortex mixing until the pellet is resuspended. Prepare a balancing tube for centrifugation. Centrifuge at 4700 g for 10 minutes at 4°C.
  3. Wash the pellet of bacteria with STE. Pour the STE wash into the original culture flask. Autoclave the culture flask with added supernatant fluid and STE wash before disposing.
  4. Several protocols using SDS (sodium dodecyl sulfate) and enzymes (lysozyme, proteinase K) are available for the lysing of bacterial cells to obtain plasmids.
  5. Resuspend the pellet in 10 mL of solution A containing 5 mg/mL lysozyme. Prepare lysozyme solution from powdered lysozyme just prior to use. Replace the cap. Vortex mix the pellet with the lysozyme/solution A mixture. Use a pipet to transfer 5 mL of the mixture to each of two Oak Ridge tubes. Maintain at room temperature. Wait for 5 minutes. Add 10 mL SDS solution (1% SDS, 0.2 M NaOH) to each tube.
  6. Cap the tube and mix the contents using a gentle rocking motion. Cool to ice temperature. Wait for 10-15 minutes.
  7. Add cold potassium acetate/acetic acid buffer to each tube. This acidifies the mixture. Cap the tube. Mix by rocking gently. Note the formation of a precipitate. Leave on ice for 10 minutes. (Bacteria are not expected to survive past this point. High levels of precaution might need to be maintained in some situations.) Centrifuge at 20,000 g for 20 minutes at 4°C.
  8. Use a pipet to transfer the supernatant fluid to siliconized Corex tubes. Divide the supernatant fluid into equal portions in those tubes. Add 0.6 mL of isopropanol for each 1.0 mL of supernatant fluid. Note the turbidity formed when the alcohol is added. Cover the tube with Parafilm® and mix. Maintain at room temperature. Wait for 15 minutes. Centrifuge at 12,000 g for 30 minutes at room temperature (not cold). The pellet contains the plasmid DNA, as well as some RNA.
  9. Carefully decant and discard the supernatant fluid. Wash with 70% aqueous ethanol. Break up the pellet. Centrifuge at 12,000 g for 10 minutes at room temperature.
  10. The ethanol-washed pellet contains the plasmid DNA, together with some RNA. Carefully decant the supernatant fluid. Touch the tube to some tissue papers to remove as much supernatant fluid as possible. Cover the tube with Parafilm®. Use a sharp needle to poke holes in the Parafilm®. Dry in a vacuum desiccator for about 10 minutes.
  11. Remove the Parafilm®. Add 1x TE buffer, pH 8.0. Redissolve the pellet. Combine fractions using a pipet. Estimate the volume of the pooled solution. Add 1 g cesium chloride for each mL of solution. Cover the tube with Parafilm®. Mix.
  12. Wear gloves -- ethidium bromide is toxic. Use a pipet to add 0.8 mL of ethidium bromide solution (10 mg/mL) per 10.0 mL of cesium chloride solution. Cover with Parafilm®. Mix. Dispose of the ethidium bromide pipet tip in a container marked for that purpose. (Treat these solutions with chlorine bleach prior to disposal to destroy the ethidium bromide.) Ultracentrifuge at 193,000 g for 36 hours at room temperature.
  13. A deeply-colored pellet and two deeply-colored bands are visible.
  14. The upper band is nicked plasmid DNA. The lower band is superhelical plasmid DNA.
  15. Wear gloves and goggles. The lower band is to be recovered. Immerse a siliconized Pasteur pipet in the tube below the dark band and withdraw that band. The withdrawn band is placed in a siliconized Corex centrifuge tube.
  16. Wear gloves and goggles. Add water-saturated 1-butanol with a disposable plastic pipet to remove the ethidium bromide by extraction. Use the pipet to agitate the phases gently. Wait for the phases to separate. Use the plastic pipet to remove the top portion each time. Discard with other ethidium bromide wastes.
  17. Wear gloves and goggles. Repeat the butanol extraction process until the aqueous phase shows no pink color. Add 3 volumes of 70% ethanol to the aqueous phase. Cover with Parafilm® and mix gently. Cool to -20°C for several hours to overnight. Centrifuge at 12,000 g for 10-15 minutes at 4°C.
  18. Decant the supernatant fluid. Wash the pellet with 70% ethanol. Centrifuge at 12,000 g for 10-15 minutes at 4°C. Cover the tube with Parafilm®; perforate. Dry under vacuum for 10 minutes. Redissolve the residue in a minimal volume of 1x TE buffer.

Going Further

The resulting solution contains a large amount of pure superhelical plasmid DNA. This might be used for transformation (A-22), restriction analysis (A-28), sequencing (B-33), or another appropriate application.

References