[admin]

Ahoy fellow researchers,

I am performing SPRI-Bead extraction of 29 day old (whole) Zebrafish.

The euthanized and frozen fish are digested for 2-5h in 20mg/ml Proteinase-K in TNES buffer.

This leaves me; depending on digestion buffer volume; with a fairly dark homogeneous lysate.

Taking a small part of this lysate and performing a down-scaled version of this protocol:

_{https://bomb.bio/wp-content/uploads/2018/09/6.1_BOMB_TNA_extraction_mammalian_GITC_v1.0.pdf}

I end up with extract that shows a very distinct gray hue.

The extraction involves GITC and several isopropanol and ethanol wash steps. After initial binding buffer removal the wash buffer always comes out crystal clear suggesting that whatever gives the final elution its colour is actively binding to the SPRI-beads.

I am assuming that the culprit is some kind of Proteinase-K + GITC resistant pigment.

Indeed it might be black eumelanin as this observation correlates strongly with individuals having developed more/less distinct dark stripes.

But, how do I get rid of it?

The obvious counter question is whether it is of any concern to my downstream sample processing. I do not know. But I would assume that if nothing else the mystery compound will occupy some of the Beads binding sites potentially leading to reduced TNA (DNA) yield.

Any ideas welcome.

Thanks everyone!!

Tim M

[Phil Oberacker]

Regarding the yield: Do you see a reduction in yield/biomass, when extracting from fish with darker stripes? And do you check the TNA(DNA) concentration with a gel or the NanoDrop? I could imagine the pigment might influence the measurement of the latter, although I don’t know the absorption of eumelanin. Also, does the pigment visibly detach from the beads during elution?

Whether or not the residual pigment influences your downstream processing I can’t say, though.

 

[admin]

Yield: I cannot say, as the fish with the darker stripes are substantially bigger at the stage I am sampling them at (because more developed). Hence, the more striped individuals give me a much more concentrated lysate when digesting the whole individual. I cannot digest only a certain volume or weight of fish and still ensure that I maintain any chosen skin (containing pigments) to flesh ratio.

As far as I am aware Eumelanin should strongly interfere with any nanodrop measurement but not dye based measurements such as is done when using Qubit.

Yes the pigments visibly detach of the beads during elution, but not the wash steps.

I have processed my samples further and I can now say that there is sufficient DNA left after PBAT. Which is/was the most important aspect. If the proportion is smaller than it would be for a pigment free sample, I have no way of telling. I guess I will just have to live with my extract looking rather questionable.

Cheers

[Tim Moser]

Ahoy, this is mainly a quick post to check whether the forum freshness display is working again. However, I figured I might as well bump this topic in the process.  Cheers
p.S.: This is Tim M’s/admin’s alias for forum functionality tests.

[Tim Moser]

Another post for the same two reasons. Cheers

[Testaccount3]

You found this post. Great. Apparently the highlight read/unread topics feature is working as long as there are no more than two levels of hierarchy (Forum>Subforum…).

 

Also, its me ,the Admin

[Author]

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