Dear all,
thanks everybody for the myriad responses I got regarding bacterial removal. Especially, Lucia, Karin, Carsten, Emily, Thiago, Shirley, and Nicole.
I am pasting here their responses so everybody can have an idea of what options we have.
Best regards,
Ana
From Karin Steffen:
–I have tried the NEB kit but for me it didn't work. As far as I remember, I didn't get any DNA in the end. I might not have done things the right way (e.g. DNA in wrong buffer/ionic strength=no binding, not enough time to release DNA from the beads). I find NEB very generous and helpful, when you ask them, they usually provide you with a free trial kit, so you could just go ahead.Maybe they also have some suggestions on which parameter to be aware of. I used their Microbial DNA enrichment kit.
–Gradients seem to work but require some (manual) practice. I tried wonce together with someone who'd done it before. I'll attach the reference she gave me.
–I'm back since this week and if you want to send me some (frozen tissue) I can try to do the extractions my way, i.e. separation by centrifugation. I tried it with Raquel's Suberites but that didn't work so well (so na guarantees... -_-"). All the dirt in it peletted as well and there was no nice layer of cells.
From Carsten Wolff:
also try Ficoll.
couple of decent (if old) papers around as you probably know e.g.:
https://link.springer.com/article/10.1007/BF02536153
http://www.science.uva.nl/ZMA/Invertebrates/Symbiosponge/rep&pub/protocols/Chapter%205.pdf
From Emily Giles:
We tried percoll gradients with centrifugation and filtering through various filters and had minimal success, especially for HMA sponges. What did work better was individually picking sponge cells with a micromanipulator though the work was tedious and in the end it was difficult to have enough cells. With few cells we had to amplify the genome which then introduces biases. Perhaps a commercial kit would work better!
From Thiago de Paula:
I was seeking a protocol for mitochondrial isolation based on cell membrane disruption (through physical strokes) followed by Percoll centrifugation. It was the best kind of protocol available for nonmodel species I could find. For me, the problem is that bacteria are usually isolated in the same density line than the mitochondria (not a personal experience though, I gave this up for practical reasons). But the nuclei partition are commonly very clean from contaminations.
You should try such organelle isolation protocols and focus on the nuclear debris instead.
From Shirley Pomponi:
Try a 10% Percoll "cushion". Play around with the percentages.
From Nicole Webster:
From our experience it seems to be entirely host species-specific so it is difficult to give one size fits all advice. We have trialled cell separations in percoll, ficoll, various manual filtrations, restriction digests etc etc (I have cc'd Sara Bell here who has done a lot of this optimisation and may be able to provide you with some of the specific protocols), however the thing that seems to have worked best for our current Ircinia species was to leave the 'dirty' DNA in water at 4 degrees for a few months. For some reason (which we have not yet managed to understand), during this time the contaminating microbial DNA appeared to preferentially degrade, leaving us with a high quality and almost pure sponge DNA preparation. It makes no real sense, but it seemed to work so you could give this a go.
