It seems to me that your annealing temperature is too low. Thus your
3 prime primer may be sticking to many non target sequences and be being
used up in non-specific transcript generation. What concentration of
primers are you using? Try using 1 or 1.5mM Magnesium. Raise your
annealing temp to 60 degrees.
I say all this because recently I have been working with a set of primers
which I designed to amplify receptor transcripts. Initially I used what
I thought were low stringent conditions ie. low annealing temp, high
Magnesium concentration and high primer concentration. Bands were weakly
amplified. Now I have switched to more highly stringent conditions, with
0.1 microMolar primers, 1 mM Magnesium and 60 degree annealing with
"touchdown" to 56 degree. Results have been spectacularly improved and
bands are now clearly present or absent in EtBr gels.
Why not give it a go?
David J. Fogarty
Department of Neuroscience,
University of the Basque Country (UPV/EHU)