Ahoy,
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
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