1. Shake E. coli harboring plasmid for 16-24 hr at 37 C in 3.3 ml of TB containing appropriate antibiotics. (when using ampicillin, add it to make final concentration of 100-200 ug/ml rather than usual 50 ug/ml. This improves the yield of plasmids.)

2. Collect the bacterial cells in a microfugte tube by spinning for 1 min at 10,000 rpm at 4 C. To collect the cells, pour 1.5ml of the culture in a microfuge tube. Spin. Discard the supernatant. Repeat this once more (or twice more).

3. (Optional) Suspend the cells in about 0.5 ml of deionized water. Spin. Discard the supernatant.

4. Resuspend the pellet in 150 ul of Solution I. Suspend the cells.

5. Add 300 ul of Solution II. Mix well by inverting the tube several times. The solution should be clear after mixing Solution II.

6. Add 225 ul of Solution III. Mix well.

7. Spin the lysate at 14,000 rpm for 1 min at 4 C.

8. Transfer the supernatant into a new tube.

9. Add 700 ul of isopropanol. Mix well. Spin at 14,000 rpm for 5 min at 4 C. Discard the supernatant.

10. Add 200 ul of 70% ethanol. Spin at 14,000 rpm for 5 min at 4 C. Discard the supernatant. Air-dry the pellet for 10 min.

13. Dissolve the pellet in 100 ul of TE containing RNaseA (20 ug/ml). Incubate for 60 min at 37 C.

14. Add 60 ul of 2M NaCl, 20% PEG8000 (PEG6000 supplied from Japanese providers is essentially equivalent to PEG8000, and works well). Mix well. Chill on ice for 5 min. Spin at 14,000 rpm for 10 min at 4 C. Discard the supernatant.

15. Add 200 ul of 70% ethanol. Spin at 14,000 rpm for 5 min at 4 C. Discard the supernatant. Air-dry the pellet for 10 min. Dissolve the pellet in 50 ul of TE. This preparation can be used for most applications including DNA sequencing. If a cleaner preparation is required, carry out step 16 described below.

16. Extract the plasmid solution with chloroform to remove PEG. Take aquaous phase. Extract the aquaous phase with phenol. Take aquaous phase. Extract the aquaous phase with chloroform or ethylether to remove trace amount of phenol dissolved in the solution. Add 0.1 volume of 3 M sodium acetate and 3 volume of ethanol into the plasmid solution. Spin at 14,000 rpm for 5 min at 4 C. Discard the supernatant. Rinse the pellet with 200 - 500 ul of 70% ethanol. Air-dry the pellet. Dissolve the DNA in 50 ul of TE.

SOLUTIONS

TB

Solution I

50 mM glucose
25 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)
Autoclave, and store at room temperature.

Lysozyme Solution

Dissolve lysozyme in Solution I (final concentration, 10 mg/ml). Dispense the solution into small aliquots (200-500 ul in each tube). Store frozen at < -20 C.

Solution II

0.2N NaOH
1% SDS
Store at room temperature in a plastic bottle. Don't autoclave!

Solution III

5M potassium acetate 60 ml
glacial acetic acid 11.5 ml
Distilled water 28.5 ml
Store at room temperature. This solution can be autoclaved. However, we usually use this solution without autclaving.

TE

10 mM Tris-Cl (pH 8.0)
0.2 mM EDTA