Triton-Prep Method for bacterial DNA Purification

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.

STET

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. 8% sucrose 5% Triton X-100 50mM Tris-HCl (pH8.0) 50mM EDTA pH 8.0 Filter sterilize. Store at 4oC