1.Grow 5 ( large scale-15ml culture). Harvest in single eppendorf tube (or 15 ml disposable tube).
2.Resuspend pellet with 300μl STET buffer (900μl). After resuspending add 30μl RNase/lysozyme mixture (100μl).
3.Boil for one minute 15 seconds (one minute 45 seconds).
4.Spin in microfuge for at least 15 minutes.
5.Take supernatant and phenol extract with 150μl (500μl) STET- saturated phenol.
6.Spin and take supernatant. Add 1/10 volume 4M lithium chloride (autoclaved). Let sit on ice for 5-10 minutes.
7.Spin and take supernatant. Add equal volume isopropanol. RT for 5 minutes.
8.Spin. No pellet will be visible. Don't panic, DNA is stuck to side all the way up tube.
9.Important: Wash with 80% ethanol (95% will cause the residual Triton to precipitate)
10.Resuspend pellet in 50-200μl.
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 -20℃ 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 4℃
