Hi there,
Trying to extract DNA from viable cells only, from a culture which is likely to contain large amount of extracellular DNA from previous biofilms and lysis.
Is there a gradient or phase separation centrifugation protocol I could apply prior to DNA extraction?
Are there kits that might do the job?
Been looking at PMA and DNA extractions as well, but the paper by Wagner et al. suggests that there might still be an overestimate of viable cell DNA coming through with this method in difficult media like sludge. I think we might run into similar issues with our extraction source.
Any other ideas?
Thanks,
Liza
I suppose you can dilute your sample and filter it then collect the cells by vortexing the cells from the filter in an isotonic solution followed by DNase I digestion to remove external DNA.
You could pretreat your culture with a nuclease, probably Benzonase would be more effective than DNase I but that might work as well. However be sure the nuclease is inactivated before lysing your cells. However the other problem you may need to consider is how do you separate the viable cells from the dead but not lysed cells.
hey Michael J. Benedik
Thanks for the suggestion - sounds like it could work. Would probably need to do some validation first, but that's ok.
Only issue I can see with this approach is that down the track most of the operators won't have an in depth micro or molecular background, which is why I thought a kit might be easier to provide training with.
Thanks again ^__^
Qiagen has a variety of kits for purifying microbial DNA from various samples. I suspect other companies do as well. You might review whether any of those meet your needs.
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