(Ali, Zaki & Clark 2005 J. Clin. Microbiol. 43: 5842-5847, modified from Clark & Diamond 1991 Mol. Biochem.Parasitol. 46: 11-18)
1. Add 250 µl of lysis buffer (0.25% SDS in 0.1 M EDTA pH 8.0) and 100 µg/ml of Proteinase K (3µl from a 10mg/ml stock) to 50 µl of culture pellet or 0.1 g of fresh or frozen stool. Vortex to disperse the cells/stool and incubate at 55° C for 20 minutes.
2. Add 75 µl of 3.5 M NaCl and mix, then add 42 µl of 10% CTAB/0.7 M NaCl (heated to 55° C), mix and incubate at 65° C for 10 minutes.
3. At room temperature, add 400 µl of chloroform, mixed well by inversion and centrifuge at full speed in a microcentrifuge for 5 minutes. The supernatant is then transferred to a fresh tube.
4. Add 400 µl of phenol:chloroform:isoamyl alcohol (25:24:1), mix well by inversion and centrifuge as above. The supernatant is then transferred to a fresh tube.
5. Add 2 volumes of 100% ethanol, mix by inversion, store at room temperature for at least 5 minutes and then centrifuge 10 minutes as above. The supernatant is discarded carefully from the pellet.
6. The pellet is washed in 200 µl of 70% ethanol by centrifuging for 5 minutes as above. The pellet is air dried and resuspended in 50 µl of sterile water (overnight at 4° C is best).
7. The resuspended DNA is then passed over a Sephacryl S200 or S400 spin column (Amersham) to remove remaining PCR inhibitors. 0.5-1 µl of this DNA solution may used in a PCR reaction.
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