I am performing in situ PCR of an RNA target in bovine lymph tissue; incorporating the DIG-dUTPs directly in the PCR product and then detection via anti-DIG Fab AP binding and subsequent incubation with BCIP/NBT.
I have used human primers on one of my controls to test for non-specific DIG staining and a lot of background staining was observed. Prior to this I performed a liquid phase PCR with bovine cDNA to ensure the human primers wouldn't produce and products, hairpins or primer dimers. There seems to be high non-specific staining in the connective tissue, but also medium non-specific staining throughout.
It has been a while since I worked with dig labeled probes, but I recall that indigenous phosphatases were often the cause of background. We added levamisole to inhibit those enzymes prior to adding the anti-dig AP conjugate. Did you try that?
Thank you David. I think that is the case in my other control that I used - no dig control, as there is some slight background staining, which could be accounted for by endogenous APs. However, in my second control using human primers there was a significant increase in background staining over and above this, suggesting non-specific dig staining?
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