Freeze/Boil Cycling for Cell Lysis for rep-PCR
Mario.Vaneechoutte at rug.ac.be
Mon Feb 19 03:48:25 EST 1996
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
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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