Method: Preparation of Miniprep Cosmid DNA from 50 ml Cultures

August 20 1990

Srini Ramachandra

Principle:

Cosmid vectors containing foreign DNA inserts are known to replicate more slowly than small plasmids in culture. One of the reasons is believed to be the capacity of the cosmids to take larger insert DNAs (up to 45 kb) causing an increased DNA replication time. Routine plasmid miniprep protocols using 5 ml overnight cultures yield very little cosmid DNA. The following protocol is a scaled-up version of the 5 ml plasmid miniprep protocol with yields ranging from 5 ug to 10 ug of cosmid DNA. This protocol is particularly helpful for obtaining higher yields of the HTY cosmids.

Time required:

2 overnight steps to amplify the cultures and half a day for DNA preparation.

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:

• GTE solution:

  stock solution            volume      final concentration
  40%  sterile glucose      2.27 ml         50 mM
  0.5 M EDTA, pH 8           2.0 ml         10 mM
  1M Tris-HCl, pH 8          2.5 ml         25 mM
  sterile ddH2O            93.23 ml
  Total:                   100.0 ml
  
  Use all sterile stock solutions. Store at 4 degrees C.

• Lysis Solution:

  stock solution          volume     final concentration
  1N NaOH	                2.0 ml          0.2 N
  10% SDS	                1.0 ml           1% 
  sterile ddH2O           7.0 ml
  Total:                 10.0 ml
  
  Prepare fresh solution prior to use.

• 3 M Potassium Acetate:

  stock solution            volume	
  5 M KOAc                    60 ml 
  glacial acetic acid       11.5 ml 
  ddH2O 	                  28.5 ml
                             100 ml
  
  Filter sterilize.  The resulting solution is 3 M potassium and 5 M
  acetate and has a pH of about 4.8.

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.