PCR doesn't work! - Falling in an empty gel "space" (Mar/25/2006 )
I've just started my project and right in the beginning I'm getting stuck trying to amplify a gene. PCR simply doesn't work. As template I'm using cDNA from a cell line in which this gene is supposed to be highly expressed. Negative and positive controls work fine. I've used computer programs to design my primers (2 sets), making sure they're not placed in the alternative splice region from the different clones from which the library is made of. Already tried betaine, DMSO, adding more Mg, ready mixes... nothing...
Does anybody have any suggestion?
have u tried changing cycling temperatures?
also, are there any other primer pairs available?
are you sure you cDNA is intact? Have you checked to see if the RT-PCR worked?