Good RT-PCR amplication but NO amplification with semi-quantitative PCR!? Wh - (Apr/17/2009 )
I am trying to look at the gene expression profiles of a certain gene during the course of an 8 day 3T3-L1 cell differentiation. I custom designed Taqman primers (from ABI) for RT-PCR and I get great amplification. The problems is that ABI does not give the sequence for which they make the primers - I only get to specify the target site of where I want the primers (i.e. between exon 2 and exon 3). Hence, in order to double check my RT-PCR results, I designed primers for conventional PCR (thus knowing the sequence). Unfortunately, I am not getting any amplification on conventional PCR (i.e. no band at all). I've tried four different primer sets, increased my cDNA in the reaction, increased the cycle number, etc and haven't gotten any bands. I'm confident with my RT-PCR results but I don't know that exactly is being amplified (since I don't know the sequence from ABI). Can anyone help me with an explanation on what to do or why this might be happening.
Ok never mind. I realized for custom ABI sequences, they do provide you with the primer sequences. My general question still stands, however, why am I not able to amplify with conventional PCR? I've tried several different primer combinations without luck.
That's odd. Are you using the same 5'and 3'primers for conventional PCR? What are you staining your gel with?
So you have verified that the ABI primers give you amplification - bands that can be seen on agarose gel. If so, does the ABI primers span an intron. I know ABI primers do not enforce this. If there is no intron and you got bands, the bands could come from genomic DNA.
How about your primers? do they span introns too?