1. Transfer a single bacterial colony into 2 ml of LB medium containing the appropriate antibiotic in a loosely capped 15 ml tube.  Incubate the culture overnight at 37 °C with vigorous shaking.

2. Pour 1.5 ml of the culture into a microfuge tube.  Centrifuge at 12.000 g for 30 s at 4°C in a microfuge.  Store the remainder of the culture at 4°C.

3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.

The supernatant can be conveniently removed with a disposable pipette tip attached to a vacuum line.  Use a gentle vacuum and touch the tip to the surface of the liquid.  Keep the tip as far away from the bacterial pellet as possible as the fluid is withdrawn from the tube.  The pipette tip can then be used to vacuum the walls of the tube to remove and adherent droplets of fluid.

Lysis by Alkali:

1. Resuspend the bacterial pellet (obtained in step 3 above) in 100 µl of ice-cold Solution I by vigorous vortexing.

2. Add 200 µl of freshly prepared Solution II.  Close the tube tightly, and mix the contents by inverting the tube rapidly five times.  Make sure that the entire surface of the tube comes in contact with Solution II.  Do not vortex.  Store the tube on ice.

3. Add 150 µl of ice-cold Solution III.  The resulting solution is 3M with respect to potassium and 5M with respect to acetate.  Close the tube and vortex it gently in an inverted position for 10 s to disperse Solution III through the viscous bacterial lysate.  Store the tube on ice for 3-5 min.

4. Centrifuge at 12.000 g for 5 min. at 4 °C in a microfuge.  Transfer the supernatant to a fresh tube.

5. Optional: Add an equal volume of phenol : chloroform.  Mix by vortexing.  After centrifuging at 12.000 g for 2 min. at 4 °C in a microfuge, transfer the supernatant to a fresh tube.

 For reasons unknown, the elimination of this step often results in DNA that is resistant to cleavage by restriction enzymes.

6. Precipitate the double-stranded DNA with 2 volumes of ethanol at room temperature.  Mix by vortexing.  Allow the mixture to stand for 2 min. at room temperature.

7. Centrifuge at 12.000 g for 5 minutes at 4 °C in a microfuge.

8. Remove the supernatant by gentle aspiration.  Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away.  Remove any drops of fluid adhering to the walls of the tube.

 The supernatant can be conveniently removed with a disposable pipette tip attached to a vacuum line.  Use a gentle vacuum and touch the tip to the surface of the liquid.  Keep the tip as far away from the pellet of nucleic acid as possible as the fluid is withdrawn from the tube.  The pipette tip can then be used to vacuum the walls of the tube to remove any adherent droplets of fluid.

9. Rinse  the pellet of double-stranded DNA with 1 ml of 70% ethanol at 4 °C.  Remove the supernatant as described in step 8, and allow the pellet of nucleic acid to dry in the air for 10 min.

10. Redissolve the nucleic acids in 50 µl of TE (pH 8.0) containing DNAse-free pancreatic RNAse (20 µg/ml).  Vortex briefly.  Store the DNA at -20 °C.

Notes:
i.   Typical yield of high-copy-number plasmids, such as Xf3 or puc, prepared by this method is about 3-5 µg of DNA per ml of original bacterial culture.
ii. To analyze the DNA by cleavage with restriction enzyme(s) remove 1 µl  of the DNA solution and add it to fresh microfuge tube that contains 8 µl of water.  Add 1 µl of the appropriate 10 x restriction enzyme.  Incubate the reaction for 1-2 hr. at the appropriate temperature.  Store the remainder of the DNA preparation at -20 °C.  Analyze the DNA fragments in the restriction digest by gel electrophoresis.
  Frequently. difficulties in cleavage with restriction enzymes can be overcome by digesting in a volume of 100-200 µl and using five- to tenfold more enzyme (especially for inexpensive enzymes).  After digestion, add 0.1 volume of 3M sodium acetate (pH 5.2) and precipitate the DNA with 2 volumes of ethanol.
iii.  The protocol can be modified to accommodate up to 10 ml of bacterial culture as follows:

a) Centrifuge 10 ml of bacterial culture at 400 r.p.m. for 10 min. at 4 °C in a Sorvall SS34 rotor (or its equivalent).
b) At step 1, resuspend the bacterial pellet in 200 µl of Solution I and transfer the suspension to a microfuge tube.
c) At step 2, add 400 µl of Solution II.
d) At step 3, add 300 µl of ice-cold Solution III.
e) At step 5, transfer 600 µl of the supernatant to a fresh tube.
f) At step 6, precipitate the DNA with 600 µl of isopropanol.

The yield of DNA is often sufficient to carry out the next step in a cloning protocol without going to the trouble or expense of a large-scale plasmid preparation.

Solution I
50 mM glucose
25 mM TrisoCl (pH 8.0)
10 mM EDTA (pH 8.0)
Solution I can be prepared in batches of approximately 100 ml, autoclaved for 15 min. at 10 lb/in2 on liquid cycle and stored at 4 °C.

Solution II
0.2 N NaOH (freshly diluted from a 10 N stock)
1% SDS (20 µl 10 N/ml, 50 µl 20%/ml)

Solution III
 5 M potassium acetate 
glacial acetic acid
H2O
 60 ml
11.5 ml
28.5 ml