I'm having problems with PCR for a few short sequences. Basically I want to identify what isoforms of the protein of interest are being expressed in the cell.
So what I've done is,
I used NCBI to design the primers by inputting the sequence I want amplified up and have double checked that it binds to the sequence.
I am using cDNA that has been converted from RNA extraction.
I have checked the Tm of the primers and they are about 50C, so according to New England Biolabs I reduced the annealing temperature from 55-->45C.
I've never really had any problems with PCR before, but this is my first time amplifying up such short sequences (50-100bp) so I'm not sure whether there are tips or tricks for dealing with short sequences. From the gel (1.5% agarose) I can see the GAPDH control band clearly at around 100+bp. So clearly the PCR method is working, but why can't I see the bands that I want?
Any advice would be appreciated.