no amplification in target gene - (Jan/08/2009 )
I am new to rt-pcr and am having some difficulties running my samples. I am trying to run the ddCt method and I get pretty good amplification curves for my housekeeping gene, but none of my target genes are being amplified. I am using the same primer and template concentrations and am using sybr green.
I don't know what I am doing wrong and if anyone has any suggestions, I would really, really appreciate it.
Thanks in advance!
I would say your target samples are pretty difficult samples. You should try optimizing their primer and MgCl concentration and the Ta also. Do they run OK in a normal conventional PCR? Are you using 1 step or 2 step realtime PCR?