Isolation of Plasmid DNA from E.coli

ISOLATION OF PLASMID DNA FROM E.COLI

Aim

  • To isolate plasmid DNA from E.coli.

Materials required

  • E.coli overnight culture, Glucose/Tris/EDTA (GTE), SDS/NaOH, Potssium Acetate, Isopropanol, Ethanol, Tris/EDTA 100 – 1000 m l micropipette + tips, 0.5 -10 m l micropipettes + tips, 1.5 ml tubes, test tube rack.

Procedure

  • Shake culture tube to resuspend E.coli cells.
  • Label two 1.5 ml tubes. Use a micropipette to transfer 1000 m l of E.coli overnight suspension into each tube.
  • Close cap, spin the tubes at 1500 rpm for 1 minute to pellet the cells.
  • Pour off supernatant from both tubes. Invert tubes, and tap gently on surface to clean paper towel to drain thoroughly.
  • Add 100 m l of ice-cold GTE solution to each tube. Resuspend pellets by pipetting solution in and out several times.
  • Add 200 m l of SDS/NaOH solution to each tube. Close caps, and mix solutions by rapidly inverting tubes five times.
  • Stand tubes on ice for 5 minutes. Suspension will become relatively clear.
  • Add 150 m l of ice-cold Pottassium acetate solution to each tube. Close caps, and mix solutions by rapidly inverting tubes five times. A white precipitate will immediately appear.
  • Stand tubes on ice for 5 minutes.
  • Spin tubes at 1500 rpm for 5 minutes to pellet precipitate along side of tube.
  • Transfer 400 m l of supernatant from each tube into clean 1.5 ml tubes.
  • Add 400 m l of isopropanol to each tube of supernatant. Close caps, and mix vigorously by rapidly inverting tubes five times. Stand at room temperature for only 2 minutes.
  • Spin tubes at 1500 rpm for 5 minutes to pellet the nucleic acids.
  • Pour off supernatant from both tubes. Be careful not to disturb nucleic acid pellets. Invert tubes, and tap gently on surface of clean paper towel to drain thoroughly.
  • Add 200 m l of Ethanol to each tube, and wash the pellet and repeat the step 2 more times.
  • Carefully draw off drops of ethanol using 1 -10 m l micropipette. Allow pellets to air dry at room temperature for 10 minutes.
  • At the end of the drying period, add 15 m l of TE to each tube. Resuspend pellets by pipetting in and out vigorously. Pool DNA/TE solution into one tube.

Questions

  • Consider the three major classes of biologically important molecules: proteins, lipids, and nucleic acids. Which steps of the miniprep procedure act on proteins? On lipids? On nucleic acids?
  • What aspect of plasmid DNA structure allows it to renature efficiently in the procedure?
Last modified: Wednesday, 20 June 2012, 6:28 AM