1. Inoculate 1 ml of L-broth or other rich media with cells of the strain from which genomic DNA is to be isolated. Grow this culture at 37°C overnight on a roller.

2. Transfer this overnight culture to a 1.7 ml microfuge tube. Pellet the cells by centrifugation on high at room temperature for one minute.

3. Resuspend and wash the cells in 1 ml of TNE buffer by pipetting gently up and down until the pellet is completely resuspended.

4. Pellet the cells by centrifuging as before.

5. Resuspend the pellet in 135 µl TNE buffer.

6. Add 135 µl of TNE buffer containing 2% Triton X-100.

7. Add 30 µl freshly prepared lysozyme (5 mg/ml). Mix well by tapping the tube.

8. Incubate in a 37°C water bath for 30 minutes.

9. Add 15 µl proteinase K solution (20 mg/ml). Mix well by inverting the tube several times.

10. Incubate in a 65°C waterbath for 2 hours.

11. Genomic DNA thus prepared may be kept at -20°C until used.

12. Digestion of the DNA is best accomplished by allowing the digestion to proceed overnight, and by keeping the volume of DNA to less than one-fifth the total volume of the digestion mixture. There is sufficient DNA in each 50 µl digestion for three electrophoretic runs on a 14.5 x 20 cm gel suitable for Southern blotting.

DNA preparation	              8.4 µl            TNE Buffer
dH2O                         34.6 µl
10X enzyme buffer             5.0 µl            Tris-HCl, pH 8.0 10 mM
Restriction enzyme (20-30 U)  2.0 µl            NaCl   10 mM
Total volume:                50.0 µl            EDTA, pH 8.0    10 mM