Plasmid isolation from gram positive bacteria
Posted 10 April 2013 - 05:17 AM
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.
Posted 11 April 2013 - 05:27 PM
Posted 15 April 2013 - 04:10 AM