1) Centrifuge 5 ml bacteria culture 5 min @ 1500g
2) Remove 1 ml broth, resuspend pellet in removed broth and transfer to 1.5 ml tube
3) Centrifuge 1.5 ml tube 30 sec @ 12,000g, dump LB, quick spin, pipet off remaining LB
4) Resuspend bacterial pellet in 200µl ice-cold solution I, vortex to completely disperse
5) Add 400µl freshly prepared solution II, invert several times, do not vortex, store on ice
6) Add 300µl ice-cold solution III, vortex inverted for 10 seconds
7) Store tube on ice for 3-5 minutes
8) Centrifuge for 5 min @ 12,000g, transfer supernatant to a fresh tube
9) Add 500µl phenol:chloroform, vortex, centrifuge 2 min @ 12,000g
10) Transfer supernatant to fresh tube with 2 volumes 100% EtOH (or until tube is full)
11) Store at room temp for 2 minutes
12) Centrifuge 5 min @ 12,000g
13) Dump EtOH, wash with 1 ml 75% EtOH
14) Centrifuge 1 min @ 12,000g, remove 75% EtOH with pipet
15) Air dry for ~10 minutes
16) Resuspend in 50-100 µl water or TE

10ml .5M EDTA (pH 8)
12.5ml 1M Tris-HCl (pH 8)
4.5g Glucose
H2O (to 500ml)
Autoclave

Solution II (10ml)

400µl 5N NaOH

500µl 20% SDS

9.1ml H2O

Solution III (40ml)

24ml 5M Potassium Acetate

4.6ml Glacial Acetic Acid

11.4ml H2O