2 replies to this topic

[watson23]

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

[susanna]

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:

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

[Mighty Mouse]

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