David,

One can use almost any extraction technique/kit that includes enzymatic digestion (Prot K) because some other ways such as guanidine lysis buffer, for instance, will not lyse egg shells of some worms, In any case, it is best to use a dedicated fecal kit or a kit that uses columns allowing to remove pigments which are strong PCR inhibitors. For instance, I found our that the standard Qiagen kit does not remove pigments very well so I used columns from another manufacturer.

Having potentially a diversity of nematode eggs in amphibian feces is a greater problem than the extract. The only way to separate later would be either having a taxon-specific set of primers or clone. The latter does not guarantee that at least some of the clones will be what you want. I would rather advocate selecting eggs. Not necessarily by flotation. Can be in sediment, just diluted enough to see through.

I used BioLine reagents long ago, but frankly, do not remember how they work.

Vasyl

Dear Vasyl,

Many thanks for sharing your experience. Actually we want to test previously developed barcodes for species collected in Herczeg et al. 2016 Comparative Parasitology 83(1):36-48. 

With previous egg selection we can increase the succes of detectability and the amount of DNA extracted, right?

Maybe I try E.Z.N.A stool DNA kit at first. It includes Protainase K. Hopefully it will be good.

Best wishes,

David

Most stool kits should work because they should take care of inhibitors. And yes, trial is likely the best way as long as your sample allows for that. Extraction from individual eggs of small nematodes is not too easy and yields low DNA, but it will be much more pure. If you will have problems please let me know and we may talk over Skype. We extracted DNA from hundreds of individual eggs and developed a few tricks for that.

Vasyl

Definitely! I'm really appreciate your kind offer. I will let you know If any difficulties appear.

Bests,

David