Protocol for Preparation of Genomic DNA from Bacteria
(Modified from Experimental Techniques in Bacterial Genetics, Jones and Bartlet, 1990)

1. Transfer 1.5 ml media to a micro centrifuge tube and spin 2 min. Decant the supernatant
2. Drain well onto a Kimwipe
3. Resuspend the pellet in 467 µl TE buffer by repeated pipetting
4. Add 30 µl of 10% SDS and 3 µl of 20 mg/ml proteinase K, mix , and incubate 1 hr at 37^oC
5. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
6. Carefully transfer the DNA/phenol mixture into a Phase Lock Gel tube and spin 2 min.
7. Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform.
8. Again mix well and transfer to a new Phase Lock Gel tube and spin 2 min.
9. Transfer the upper aqueous phase to a new tube.
10. Add 1/10 volume of sodium acetate.
11. Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates.
12. Centrifuge to pellet DNA.
13. Wash in 70% Ethanol.
14. Resuspend DNA in 100-200 µl TE buffer.
15. After DNA has dissolved, measure the concentration by diluting 10 µl of DNA into 1 ml of TE (1:100 dilution) and measure absorbance at 260 nm.
16. Concentration of original DNA solution in µg/ml = Abs x 100 x 50 µg/ml.

Caution: Phenol causes severe burns! Wear gloves, goggles and lab coat! Keep tubes capped tightly.