Designing Primers for RT-PCR after ChIP - Help to avoid primer dimers (Oct/23/2009 )

Hi,
I am new to this science forum and I signed up to see if anyone had any ideas on overcoming a primer problem.

I am designing primers for RT-PCR (after ChIP) for two Sp-1 sites on my target gene promoter. These two sites are so close together on the promoter that it's impossible to design two individual primers - so I need one primer for the two sites.

I am having trouble finding primers that don't make self-dimers or hairpin structures due to the high C/G content and the fact that this primer is longer to cover both Sp-1 sites.

I have tried reducing the CG content, primer length, probe length, number of C/G at 3' and 5' ends, amplicon length - on the parameters for the Primer Express program that I use.

The primers have been re-ordered twice already, both giving me obvious dimers in their melt curves and efficiency of around 85% for the standard curves. I have also changed the concentration of the DNA template and the primer concentration but the dimers are still present.
I was wondering whether anyone has designed one primer for two sites, or any ideas for things to try?

Thank you in advance
Amelia, Belgium

you can check if your primers make primerdimer by programs as "Oligo 7-software"
But usually your primers you get with primer-blast are quite ok, too
you can also test them in the experiments, by calculation of the melting curves

watson23 on Oct 23 2009, 04:59 AM said:

I've had better luck using Primer3 (http://frodo.wi.mit.edu/primer3/) for primer design than the Primer Express program offered by ABI. You can tweak more of the parameters with Primer 3, which is nice. I always follow up with analysis using Net Primer for self/cross dimers and hairpins, then BLAST...

MM