Gram-positive or Gram-negative bacteria? For Gram-negative, you don't need a lysozyme treatment.

Gram positive (staph aureus)

Suggest looking at this link:

http://richardsonlab.web.unc.edu/protocols/colony-pcr-for-s-aureus/

Hey Ian,

I am currently working with Bacillus and I used exactly the first protocol on the attached link, just changing proteinase k for the lysosyme I had left from a bacterial DNA extraction kit (Himedia). Although this method does cause DNA fragmentation, depending on the size of your target, it shouldn't be a problem to amplify.

I performed PCR for the 16S rDNA (~1.5 kb) with the lysate without any further purification. I just had to centrifuge the samples in cases where there were some debris left.

Best luck!

http://www.uni-tuebingen.de/fileadmin/Uni_Tuebingen/Fakultaeten/Biologie/Institut_fuer_Evolution_und_Oekologie/Evolutionsoekologie_der_Tiere/Dokumente/research/methods/DNA_Isolation_180706.pdf

Using a fresh culture, you could try resuspending a few colonies in 150 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA), boiling at 98ºC during 10 min in the thermocycler and using the supernatant as DNA template (5 µl) for amplification.  It works well with Bacillus anthracis.