SSCP - (Feb/04/2010 )
I've been trying for some time to complete SSCP, but still haven't succeeded til the very end. The problem is that everything goes almost as it should be, but in the end I never get the zones to show up, the gel itself is in the necessery colour, but even gene ruler can't be seen. Has anyone tried to use this method and also had some problems? What problems could there possibly be? I've gone through the protocol over and over again, but still don't know what could be the reason for this.
Do you know for sure, that your PCR reactions were successful? Make sure your math for mixing the stain is correct. Make sure you are using fresh staining solution. When I did SSCP using silver nitrate as my stain, I would reuse the stain solution only a few times before making it fresh. But that all depends on how concentrated a solution you use.
Yes, I'm absolutely sure, that PCR was successful and even if it wouldn't be, still, I would have to see the ruler. and about the solutions I make them always just before the staining. The gel always turns yellow as it should be, but the zones don't show up. The strange thing is that I can see that the electrophoresis is working, 'cause the stains are migrating, so I can't understand, where the products disappear.