We are working with Gram-positive soil actinobacteria (Frankia). There are several methods to lyse Frankia cells in order to extract their DNA. But now we have a Frankia strain - not culturable, isolated from nodules - on which these methods (enzymatically with lysozyme and achromopeptidase, mechanically with FastPrep, repeated freeze/thaw) simply do not work (yes, we do controls, and yes, the methods work on other strains). Does anybody have an idea what we could try? We have very limited amounts of material - this failure to lyse struck us out of the blue...
1-You can use bead beater (Mechanical Method) for DNA isolation...http://www.mpbio.com/product.php?pid=116004500&country=162
With this method DNA product is less contaminated with chemicals So we can easily amplified these DNA as compared with other method.
2-Grind nodule with liquid nitrogen and try sonication after applying lysozyme...
Its time for good old mortar pestle using liquid nitrogen, you get good quality lysis with minimal degradation.I do not understand the part --not cultivable but isolated from nodules..can you please elaborate on that ?
Jay Siddharth: We can isolate the bacteria from the symbiotic plant organs. We cannot persuade the bacteria to propagate outside of the symbiotic plant organs - i.e., we cannot culture them. So for practical purposes, we are working with an obligate symbiont. (Of course, maybe it's not an obligate symbiont, only nobody ever tried the right culture conditions).
Katharina, thank you very much for the insight.
I think the liquid nitrogen method would work, I would be interested to know how it goes, do keep me posted.
Cheers
JS
depending on my experience think ultrasonication in chilling conditions is the technique of choice for lysis of Gram positive, and thus not constitute a burden and a loss of time in the disposal of subsequent steps
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