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I have been trying to immobilize DNA by the bioconjugation of biotin and streptavidin, but I cannot get this work.

I added EDC and streptavidin to COOH functionalized resin, reacted for 3 hours in room temperature, and then washed with PBS.

Then I tried to anneal biotin-labeled primer and FAM-labeled complementary sequence. (Heated it up to 95 degree then let it cool down to room temperature.

After I added SA-functionalized resin and FAM-Biotin strands together, the conjugation wouldn't work. The resins are not fluorescent after wash.

I also tried to use PCR products which are labeled on both side.

Is there any problem with my assay? How long it takes for the conjugation of biotin and SA at very low concentration?

Thank you for any information.

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  • $\begingroup$ Are you certain the resin has streptavidin? Can you get some biotinylated fluorescein or other dye just to check the streptavidin? HABA dye might also be helpful to determine if streptavidin is bound. $\endgroup$
    – user137
    Jan 30 '15 at 15:57
  • $\begingroup$ @user137 Thank you for your advice! I am not sure if resin is successfully functionalized, as I don't have any dye to check for now. Maybe I should purchase one for this. I don't see any reason that SA is not bond, I saw a lot of paper use this method. $\endgroup$
    – leetom
    Jan 30 '15 at 16:53

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