I will need to isolate low copy plasmid from L. lactis to amplify a specific region of it later on.It will be my first time working with G+ bacteria. We have several miniprep kits in the lab, but those are optimized for E.Coli. I read in an other kit's protocols (I can't use it bc that one is good for genomic DNA isolation) that I should treat the cells with lyzosime. I was thinking about treat the cells with lyzosime first and then isolate the plasmids with the help of the miniprep kits in our lab. But I have met with different obstacles for example what should be the stock of the enzyme solution etc. What is the most efficent way/protocol to isolate plasmid form L. lactis ? Thanks for your help/tips in advance!
Hi Henrietta,
You could resuspend L. lactis cells in about 30-50 µL of water and heat this suspension to about 100 °C for 10 minutes. The resulting solution should be cleaned by centrifugation (e.g. one minute at full speed). Afterwards, the supernatant can serve as template for PCR. It is a very simple method that worked for the amplification of chromosomal sequences in my hands. If you are interested in plasmids, it should work even better.
Good luck!
I used either Qiagen or Promega miniprep kit, not Thermo, it doesn't work. I use the protocol but adding an extra step. At the beginning when you resuspend the pellet I add 50 ul of lisozime 50 mg/ml and I incubated 1h at 37ºC. Then I go on with the protocol.
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