Hi All,
I’m trying to learn to do DNA-PAINT following the Jungmann Nature Protocols paper. I’m using Ibidi 6 well flow chambers. I’ve noticed that the imager strand conjugated to Cy5 is very sticky and creates a lot of background, while a similar sequence with Cy3 does not.
Does anyone have recommendations for cleaning the slide or changes to the buffer that would prevent Cy5 from sticking to the glass? Since I want to use these pre-made chambers for convenience, I don’t think plasma cleaning will be feasible (nor do I have that equipment).
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Hi Sara,
it has been a while since I have been imaging with Cy5 so I can not really recall right now if there was a a lot of non-specific binding to the surface or a high background. In any case I think the Cy5 dye might not be the best dye for DNA-PAINT. I would recommend switching to Atto647N or Atto643 which both worked well for me. I further more would recommend using Trolox, PCA and PCD (you could also try to combine this with your current Cy5 imager it should also boost the performance of the Cy5 dye) as described in our protocols paper.
For the green excitation channel I would also recommend to switch from Cy3 to Cy3b.
I hope that helps!
Best wishes from Munich,
Florian
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