Freeze/Boil Cycling for Cell Lysis for rep-PCR

Mario Vaneechoutte Mario.Vaneechoutte at rug.ac.be
Mon Feb 19 03:48:25 EST 1996


Dear Lemuel,

We've been trying and comparing different simple DNA-extractions 
of bacteria for PCR since three years, with a lot of trouble. Since three 
months we are using alkaline lysis and things work perfect now for any 
kind of PCR-based DNA-fingerprinting we do.

Pick a small (1-2 mm) colony into 20 microliters of lysis buffer (0.05 M 
NaOH/0.25%SDS) and heat for 15 min at 95 degrees. Dilute with 200 µl of 
sterile water. Spin down cell debris for 5 min. Use 1-2 µl for a 25 µl 
PCR volume.

In our hands this works for gram+ , gram- and mycobacteria.

To avoid problems with NaOH precipitation during storage we use a 
commercially prepared 1 M NaOH solution (Merck N° 109137). The NaOH-SDS 
mixture is stored in plastic tubes (not glass). The SDS precipitate which 
forms is easily resolved by heating in a microwave for a few seconds.

Let me know whether this works for you too. Success.




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