2 replies to this topic

[2xzwei]

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.

[phage434]

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

[2xzwei]

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.