Rhodobacter genomic DNA extraction protocol.
(1988, Mol Gen Genet, 213, p78-83)
- pre-heat buffer to 65°C
1) Pellet 1.5 ml of stationary phase culture in a microfuge tube (for most centrifuges a
1min spin is enough). Discard supernatant.
2) Freeze tubes in liquid nitrogen.
3) Gently resuspend pellet in 0.5 ml of 65°C lysis buffer (10mM Tris pH8, 1mM
EDTA, 1%SDS). The buffer should be preheated to 65°C in a water bath prior to use.
4) Add 100 g (10ul) proteinase K (stock: 10 mg/ml in sterile distilled water, store at
–20°C) and incubate the tube at 45°C for 2 hours.
5) Extract twice with 0.5 ml of phenol-chloroform-isoamyl alcohol (25:24:1) in
eppendorf tubes containing silicon grease – shake well to mix and spin for 5mins.
Pour top layer into new phenol/grease tube, shake and spin.
6) After the last phenol extraction, pour the top layer into 1 ml of Ethanol to
precipitate the DNA. Mix and leave at -20°C for 30mins.
7) Spin for 15 min and then wash once with 1 ml of 70% ethanol. Spin for a further 15
mins. Dry the pellet overnight at 37°C.
8) Resuspend the pellet in 50 l of water and store at -20°C.