PLASMID QUICK PREPS

In a little over one hour, this procedure yields enough DNA for 10-20 gel lanes from a 5 ml overnight saturated bacterial culture. This is a modification of the technique of Blin & Doly in Nucleic Acids Research 7:1513-1523 (1979).

Procedure
  1. Grow a 10 ml overnight bacterial/plasmid culture in selective media. Freeze half as a bacterial stock and use the other half in this protocol.
  2. Prepare in advance per 5 ml culture: Two 1.5 ml microcentrifuge tubes with 0.5 ml isopropanol in one; Solution I (180 µl); Solution II (400 µl, not more than one we ek old); Solution III (300 µl, keep cold in a refrigerator); Lysozyme solution (20 µl, 5 mg/ml in solution I).
  3. Centrifuge the cells at 2500 rpm for 12 minutes at 4°C and carefully pour off the supernatant.
  4. Resuspend the cells in 180 µl of solution I and transfer to an empty microcentrifuge tube. Add 20 µl of lysozyme solution and give it a quick vortex. Incubate for 5 minutes at room temperature.
  5. Add 400 µl of solution II. Mix by invers ion a few times - do not vortex. Incubate on ice for 5 min.
  6. Add 300 µl pre-cooled solution III. The tube contents should be very viscous. Mix with a quick vortex. Incubate on ice for 10 minutes.
  7. Centrifuge the sample for two minutes in a microcentrifuge at room temperature, then carefully transfer the supernatant to a second microcentrifuge tube containing 500 µl (0.6 volumes) of isopropanol to precipitate the DNA (avoid transferring the white material). (Optional: To increase the yield - centrifuge the tube containing the residual white crud for another two minutes, collect any supernatant, and add it to the above supernatant.)
  8. Vortex and centrifuge for 3 minutes in a microcentrifuge at room temperature. Pour off as much supernatant as possible. At this point, you should be able to see a small white pellet in the bottom of the tube.
  9. Resuspend the pellet in 196 µl of TE. Optional phenol and ether extractions should be performed at this poin t if you intend to save the DNA for more than about a week. Dissolve pellet by vortexing, then add 4 µl of 5M NaCl and 0.6 ml of cold 95% (or absolute) ethanol. Precipitate as usual. Spin, dry, and dissolve pellet in 50 µl TE.
  10. 3-6 µl of this quick prep DNA per gel lane. The DNA is efficiently digested with a variety of restriction enzymes. The large amount of RNA present in the sample may be digested by adding 1 µl of a 250 µg/ml stock of RNAse for 2 minutes a t room temperature at the end of this digestion. (It should be added if you'd like to see DNA < 1.5 Kb). You can roughly estimate the amount of DNA by doing a 1/10 to 1/50 dilution of the DNA in TE, adding 1 µl of the above RNAse for 2 minutes at room temperature, and spotting a sample on an ethidium bromide plate versus plasmid DNA standards (degraded RNA will not stain on ethidium bromide plates).
RECIPES

Solution I:   50 mM Glucose (0.9% w/v); 25 mM Tris pH 8, 10 mM EDTA pH 7.5

0.9 g Glucose
2.5 ml 1.0 M Tris
2 ml 0.5 M EDTA
QS to 100 ml with water
Solution II:   0.2 N NaOH, 1% SDS
105 µl 50% (w/w) NaOH stock (or 200µl 10N NaOH)
1 ml 10% SDS stock
8.9 ml water
...KEEP AT ROOM TEMPERATURE... KEEPS ABOUT 1 WEEK
Solution III:  2.7 M KOAc to pH 4.8 with glacial HOAc.
26.5 g potassium acetate in minimum water then pH.
q.s to 100 ml with water.
KEEP IN REFRIGERATOR
Lysozyme
5 mg/ml made fresh in Solution I.
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Plasmid Quick-Preps. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/quikprep.html). 1997. Oklahoma City. Revision Date: October 2, 1997."