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Method: Preparation of Miniprep Cosmid DNA from 50 ml Cultures

August 20 1990

Srini Ramachandra


Principle:

Time required:

Procedure:

Day 1

    At the end of the day start a 5 ml overnight culture of each cosmid in LBM medium containing the appropriate antibiotic. Use a freshly streaked single colony (not more than a week old) for inoculating the 5 ml medium and incubate the culture tubes at 37 degrees C on a roller drum.

Day 2

    Inoculate 100 ul of the overnight culture into a 125 ml sterile glass flask containing 50 ml of LBM containing the appropriate antibiotic and incubate overnight in a 37 degrees C shaker incubator with vigorous shaking (~ 200 rpm).

Day 3

  1. Transfer the 50 ml cultures to 50 ml orange screw-cap tubes and spin the tubes in the Beckman J-6 centrifuge at 2500 rpm 15 minutes at 4 degrees C.
  2. Discard supernatant and resuspend pellets in 2 ml of GTE solution. Incubate at room temperature for 5 minutes.
  3. Add 4 ml of freshly prepared Lysis solution. Mix gently by inverting the tubes and place on ice for 5 minutes.
  4. Add 3 ml of ice-cold potassium acetate solution (3M potassium 5M acetate) and mix gently. Invert the tubes and vortex gently for 10 seconds. Place on ice for 5 minutes.
  5. Spin the tubes in the J-6 centrifuge at 2500 rpm 5 minutes at 4 degrees C. Transfer the supernatants gently to new tubes taking care not to disturb the white pellet (which contains proteins RNA and E. coli chromosomal DNA). Discard the pellet.
  6. Add an equal volume (approximately 7 - 8 ml) of phenol/chloroform mix. Mix thoroughly and spin the tubes in the J-6 centrifuge at 3000 rpm 10 minutes at room temperature.
  7. Transfer the aqueous (top) phase (approximately 7 ml) to new tubes and add 0.6 volumes (approximately 4.2 ml) of cold isopropanol. Mix well and incubate for 15 minutes at room temperature. Discard the phenol/chloroform phase into the phenol/chloroform waste container.
  8. Spin the tubes in the J-6 centrifuge at 3000 rpm 15 minutes at room temperature. Discard the supernatant. Wash the pellet with 5 ml of 70% cold ethanol. Spin at 3000 rpm for 15 minutes at room temperature in the J-6 centrifuge.
  9. Discard supernatant and repeat ethanol wash as in step 8.
  10. Drain the supernatant and air dry pellets on the bench top for at least 2 hours. Resuspend pellets in 500 ul of TE (pH 8.0) containing 0.02 mg/ml RNAase A) and incubate at 37 degrees C for 30 minutes. Run a 10 ul aliquot of the DNA on a 0.8% TA-agarose gel against concentration standards to check for purity and yield.

    Note: If a pellet does not resuspend easily in step #10 let it sit at 4 degrees C overnight to aid resuspension.

Solutions:

References:

Sambrook J. Fritsch E.F. and T. Maniatis (1989). Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp.1.25-1.28.

Harold Riethman pers. comm. @ cosmid miniprep protocol.