Triton-Prep Method for bacterial DNA Purification
- Grow 5 ( large scale-15ml culture). Harvest in single eppendorf tube (or 15 ml disposable tube).
- Resuspend pellet with 300ul STET buffer (900ul). After resuspending add 30ul RNase/lysozyme mixture (100ul).
- Boil for one minute 15 seconds (one minute 45 seconds).
- Spin in microfuge for at least 15 minutes.
- Take supernatant and phenol extract with 150ul (500ul) STET- saturated phenol.
- Spin and take supernatant. Add 1/10 volume 4M lithium chloride (autoclaved). Let sit on ice for 5-10 minutes.
- Spin and take supernatant. Add equal volume isopropanol. RT for 5 minutes.
- Spin. No pellet will be visible. Don't panic, DNA is stuck to side all the way up tube.
- Important: Wash with 80% ethanol (95% will cause the residual Triton to precipitate)
- Resuspend pellet in 50-200ul.
- Lysozyme/ RNase mixture
- 10mg/ml lysozyme
- 1mg/ml RNase (use cheap grade (BMB) rather than RNase A , which is too expensive)
- 50mM Tris-HCl pH8.0
- Store at -20oC in small aliquots. Do not refreeze after thawing.
STET
- 8% sucrose
- 5% Triton X-100
- 50mM Tris-HCl (pH8.0)
- 50mM EDTA pH 8.0
- Filter sterilize. Store at 4oC
- 1mg/ml RNase (use cheap grade (BMB) rather than RNase A , which is too expensive)
