Triton-Prep Method for bacterial DNA Purification

 

Triton-Prep Method for bacterial DNA Purification

1. Grow 5 ( large scale-15ml culture). Harvest in single eppendorf tube (or 15 ml disposable tube).

    

2. Resuspend pellet with 300ul STET buffer (900ul). After resuspending add 30ul RNase/lysozyme mixture (100ul).

    

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 150ul (500ul) 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 50-200ul.

     

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