I have a question for the professionals. The essence of the problem: I fix the oligos on a plastic substrate, they play role of primers in solid-phase PCR. I carry out a one-step PCR with simultaneous labeling of the product with biotin (I add 10% labeled uridine to unlabeled T to the DNTP mixture), then I wash it in PBS and incubate it with the streptavidin-peroxidase complex and then incubate it with the substrate for peroxidase. Everything would be fine, but in the control wells, where the PCR reaction mixturedoes not contain DNA, I have a staining of oligo spots, weaker than in the experimental wells, but it is there. Moreover, in the control wells, where only PBS was added, weak staining also appears at the localization spots of the oligos. If I simply add the complex to the wells (without any PCR treatmen), then only the positive control points, that is, the initially labeled oliagos, are stained in them. So here's the question. Can streptavidin (or peroxidase) bind to something other than biotin or DNA oligos? I’ve been fighting with the problem for a couple of months now. I 've changed blocking buffers, polymerases, washing modes, but the result is still the same. Help, good people!
The streptavidin-biotin bond is very specific, but the biotinylation is never 100% efficient. Might try to pre-select biotinylated oligos only prior to the streptavidin step
Hi, Peter. Thanks a lot for your answer to my question. I did all possible combinations of PCR components, and I added two additional controls with PBS and water only in wells. But, after incubation with streptavidin-peroxidase complex and substrate for peroxidase I've got the stainig even in control wells. There was nothing in these wells, only PBS and water. It looks like, streptavidine or may be peroxidase somehow bind to oligos immobilized on plastic slides. This staining is weak, but it is there. I can't explaine this.
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