Method: Alkaline Lysis Minipreps of Plasmid or Cosmid DNA

May 1 1990

C. Helms

Principle:

Plasmid or cosmid containing E. coli cells are grown and lysed with alkali. The cell debris and chromosomal DNA is precipitated with SDS and potassium acetate. After pelleting the debris the plasmid/cosmid DNA is precipitated from the supernatant with isopropanol and the DNA resuspended in TE. The expected yields from a 5 ml culture are 5-10 ug for plasmids 2-5 ug for cosmids. The procedure can be scaled up many fold.

Special solutions required:

• GTE solution

• lysis solution

Time required:

approximately 2 hours of benchwork

Procedure:

Day 1

1. Inoculate 5 ml of LBM medium containing 50 ug /ml ampicillin (use 200 ug / ml for cosmids) with a single bacterial colony containing the desired plasmid or cosmid. Incubate at 37 degrees C overnight on a roller drum.

Day 2

1. Spin down cells at 2500 rpm in Beckman low speed centrifuge (e. g. the J-6) for 15 minutes. Discard supernatant.

2. Resuspend each pellet in 200 ul GTE solution. Transfer samples to labeled eppendorf tubes. Incubate at room temperature for 5 minutes.

3. Add 400 ul of a freshly prepared solution of lysis solution. Mix gently. Place on ice for 5 minutes.

4. Add 300 ul of an ice-cold solution of 3 M potassium acetate (see recipe at end of procedure). Mix gently. Place on ice for 5 minutes.

5. Centrifuge for 1 minute at 4 degrees C. Transfer the supernatant to a clean tube.

6. Add 0.6 volume (540 ul) isopropanol to the supernatant mix and incubate at room temperature for 2 minutes. Pellet the nucleic acid (a 1 minute microcentrifuge spin top speed). Discard the supernatant.

7. Wash the pellet twice with 1 ml 70% ethanol. Spin for 1 minute and discard the supernatant.

8. Dry the pellet for 10 minutes in the lyophilizer or allow to air dry. Resuspend in 50 ul TE pH 7.5. Add 1 ul of a 10 mg/ml solution of RNAase and incubate for 30 minutes at room temperature.

9. Assay the DNA on a minigel with appropriate concentration standards before restricting the DNA. The expected yield is 5-10 ug plasmid DNA or 2-5 ug cosmid DNA from 5 ml of culture.

   NOTE: This procedure can be scaled up many fold. For cell culture volumes over 25 mls reprecipitate with isopropanol after step 9 since a great deal of KOAc is still held in large pellets (which lowers pH). Before banding the DNA in CsCl check that the DNA solution is of neutral pH.

Solutions:

1. 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.  

2. 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. 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.  

4. RNAase (10 mg/ml):

       Dissolve 100 mg RNAase A (pancreatic RNAase)    in 10 ml 10mM Tris-HCl 15 mM NaCl.    Heat to 100 degrees C (in a beaker of    boiling water) for 15 minutes.  Cool    slowly to room temperature. Dispense into    aliquots and store at -20 degrees C.  

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.