This procedure was originally developed for Listeria monocytogenes but has worked well with other Gram+ bacteria we've tried.

1.Pellet cells from 10 ml overnight cultures in BHI or LB and wash in 5 ml of 0.1X SSC.

2.Resuspend in 1 ml 10 mM Tris-HCl (pH 8.0)containing 20 % sucrose (v/v),add lysozyme to 2.5 mg/ml,and incubate at 37℃ for 45 min.

3.Add 9 ml lysis buffer (10 mM Tris-HCl [pH 8.0],1 mM EDTA,500 mg pronase B,1 % SDS),and incubate additional 30 min at 37℃.

4.Phenol and chloroform extract lysed cells,and ethanol precipitate the DNA with 0.1 vol.3 M sodium acetate,pH 4.8 and 2 vol.95% ethanol.

5.Spool out DNA with a glass rod,wash once with 80% ethanol before drying.

Some bacterial species may require a longer incubation in lysozyme.For Renibacterium salmoninarum (a G+ salmon pathogen we work with),lysozyme incubations overnight at 37℃ worked very well with high yields of DNA

ref: Flamm,R.K.,Hinrichs,D.J.,and Thomashow,M.F.1984.Introduction of pAM 1 into Listeria monocytogenes by conjugation and homology between native L.monocytogenes plasmids.Infect.Immun.44:157-161.