I hope to extract the DNA of Lactic acid bacteria (LAB) for quantitative analysis.
I have put lysozyme 50 ul (60 mg/ml), RNase 3 ul (1 mg/ml), 25% SDS (25 ul)
and proteinase K (25 ul) after washing the cultured broth with TE buffer.
However, concentration of Leuconostoc citreum is under 2 ng/ul.. its too low.
Adding mutanolysin is better?
then I want to know its concentration and volume.
Also,,, Is there any other method? :-)
observe the cell lysis step ... do not proceed if you do not have a clear solution
may use bead beating...
Hi
You can use promage's Kit of Gram positive bacteria. It's very best for DNA extract from lactic acid bacteria .
I used the protocol you found in attach file
Centrifuge 4 ml of culture and resuspend pellet in 10 ml of fresh medium (MRS broth is preferred). Incubate for 2 hrs at 37°C
- Centrifuge again and resuspend pellet in 1 ml of cold 25% sucrose, 0.05 M Tris, pH 7.5, 5 mM EDTA at 4°C
- Keep cell suspension in ice bath for 10 min, then add 75 µl of lysozyme (1 mg/ml in 0.05 M Tris, pH 7.5, 5 mM EDTA), mix and incubate in ice bath for 1hr (for some
strains incubation at 37°C for 1hr is preferred)
- Centrifuge cells and add 500 µl of the following lysis solution to the pellet and mix well: 0.05 M Tris, 5 mM EDTA, 0.05 M glucose, 3% SDS.
- Heat the sample at 62°C for 1 hr, then allow to cool slowly (approx.15 min) to RT.
- Follow PCI method for DNA extraction.
Hope this helps.
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