Miniprep method

jeff jeff
Tue May 13 16:35:15 EST 1997


On Tue, 13 May 1997 17:53:43 +0100, jmejia at worf.molbiol.ox.ac.uk (Jose
E. Mejia) wrote:

>I am looking for protocols or kits fo plasmid minipreps that do not
>involve denaturation of DNA, i.e. neither alkaline nor boiling lysis. If
>anyone knows about any such methods, please drop me a line.

my favorite:

1. Grow cells overnight in LB at 37 C with shaking.
2. Microfuge cells for 30 sec.  Remove supernatant by aspiration.
3. Resuspend cells in 100 uL TELT Buffer: 

50 mM Tris, pH 8.0
62.5 mM EDTA, pH 8.0	
2.5 M	LiCl
4 %	Triton X-100

4. Add 100 uL 25:24:1 Phenol/Chloroform/Isoamyl alcohol. 
5. Vortex vigorously for 15 sec.
6. Centrifuge for 1 min at room temperature.
7. Transfer supernatant to a new microcentrifuge tube.  
8. Add 200 uL 95% Ethanol.  Mix well and incubate at room temperature
for 2 min.
9. Microfuge for 5 min at 4 C.
10. Discard supernatant.  Add 1 mL 70% Ethanol, swirl, and discard.
11. Air dry.  
12. Resuspend in 30 uL TE.


Jeffrey Lawrence
Dept of Biological Sciences
University of Pittsburgh
Pittsburgh, PA 15260



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