We´ve already tried different kits (PowerBiofilm and PowerSoil (MOBIO) and DNeasy Blood and Tissue kit (QIAGEN)) but we are not able to get measurable DNA (we´ve tried both NanoDrop and PicoGreen assay). Could anyone give me any suggestion if this could be due to very low starting material on skin swab (how to deal with it?)? Or due to wrong procedure?
Before taking swab sample we premoistured it in SCF-1 buffer (50 mM Tris buffer, 1 mM EDTA, 0,5% Tween20). Took sample, snapped off swab into bead beating tube, added lysis buffer (usually recommended by manufacturer), incubated 30min / 50-60°C (with QIAGEN overnight), bead beat sample (with swab inside) and followed manufacturers´ protocol.
I´d be very grateful for any advise how to get better yields of bacterial DNA from skin swabs.
Thank you very much!
Zuzka
Have you tried subculturing before DNA prep?...for instance, simply plate out all bacteria isolated from skin in buffer collection on rich media. Separating bacteria from Yeasts with selective media. Don't know if this would help if you are trying to "shotgun" this to get rapid results, but sometimes old tried and true Microbiological techniques actually save you time in the long run...Hope this helps you...
Hello Christopher,
thank you for your suggestion. Subculturing I didn´t try. We are interested in "any single bacteria" (its DNA) from the skin, we´d like to use pyrosequencing to identify microbial population of the skin. I am not sure whether I can subculture bacteria from swabs, because I am afraid to loose unculturable or fastidious species which could be overgrown for example by common and fast-growing staphylococci...Thank you very much, I appreciate you found time to try to help me.
Kind regards!
I haven't used it myself, but some colleagues have successfully used the Qiagen AllPrep kit on sinus swabs.
https://www.qiagen.com/nz/shop/sample-technologies/rna-sample-technologies/dna-rna-protein/allprep-dnarna-mini-kit/
Article The nasal microbiota in health and disease: Variation within...
Hi take full loop from colonies of pure culture of bacteria under interest ,dilutes with 0.5 ml SDW and heat for 10 mints at 90 C let the tube be cool , add 200 µL chloroform-phenol to the cells lysates and mixed by vortex for 1 min. centrifuge for 2min .trans the supernatant to new tube and add 700 µL of absolute ethylalkhol and mixed with vortex and centrifuge for 12min at 13000 rpm. .evaporate and dry and load 8 micro liter DNA with load stain and electrophoresis
Hello David and Zaidan,
thank you both for your suggestions!
Best regards
Try the new PureLink Microbiome DNA Purification Kit – it has protocols for swabs etc http://www.thermofisher.com/order/catalog/product/A29790?ICID=search-product
Hi Zuzana,
I am trying to isolate the skin bacterial DNA from swab/buffer scrub samples. I am facing a similar problem like you. Could you please suggest what you have done to overcome your problem. In my case, I have to directly extract sample DNA and take it for analysis, similar to what you have done. I tried using MoBio Power soil and also Lysozyme followed by Blood & tissue QIAGEN kit protocol. Nothing has worked. Please share your learnings.
Thanks in advance
Bharat
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