Plasmid isolation from gram positive bacteria - (Apr/10/2013 )

Hi there!

Currently I'm struggling with the isolation of a plasmid from Enterococcus faecalis (gram positive bacteria). In a first run I tried it by following the standard protocol for plasmid extraction (Qiagen kit) but I didn't yield any plasmid. So I read about the addition of Lysostaphin which should crack the murein layer (0.5mg/mL 10µL per 250µL suspension) and incubated it for 45min @37°C. But this didn't help, too

Now I tried the addition of Lysozyme (in Tris-HCl buffer pH 8) to the suspension buffer (100µL of 10mg/mL Lysozyme stock) and incubated 30min @37°C. But all I got was some smear on my agarose gel and no plasmid...

Does anyone of you have further suggestions how to successfully isolate plasmid from gram positive bacteria (esp. E. faecalis)? I read about methods with heat-lysing (heating to 80°C for a while) but I couldn't find any protocol for that...

Thanks in advance for your help!

PS: Yes the plasmid is inside, otherwise it wouldn't grow on my antibiotic agar.

Often gram positive strains will integrate plasmids into the genome. If that happened, you'll have trouble isolating it.

I finally got it work. The Lysozyme I initially used was old. Ordered a new one and repeated the isolation process and it worked quite fine.