What are the most important parameters in PCR primer design? - Tm difference, GC clamp, GC%, primer-primer complementarity, self-complementarit (Dec/05/2006 )

I would like to sap your PCR experience? I'm wondering of all the various parameters that you can set while designing your PCR primers which are the most important? Do you believe in GC clamps? And of which length? To you subscribe to very low Tm differences between primers? Do you scrutinise your primers for hours to find complementarity?

Do you know any publications or web sites that address this issue?

Hope to hear from you PCR-savy people,

~j

I would like to sap your PCR experience? I'm wondering of all the various parameters that you can set while designing your PCR primers which are the most important? Do you believe in GC clamps? And of which length? To you subscribe to very low Tm differences between primers? Do you scrutinise your primers for hours to find complementarity?

Do you know any publications or web sites that address this issue?

Hope to hear from you PCR-savy people,

~j

Hmm, I wanted the poll to accept multiple answers but that doesn't seem to work. Is there a way to do that or does the forum software just not allow for that?

~j

i vote for

small Tm diferences between primers (e.g. 1-2°C max.)
no nucleatide repeats (e.g. ...AAA..) more then 4. G repeats are the worse
small primer-primer complementarity
small self-complementarity

my primers must fit all these parameters. Failure = rejection.

P.S: I do not scrutinise my primers for hours on end for complementarity... I use a programe to do that for me

18-22 bp long, G or C in 3' position, near 50% GC, lower better than higher, no hairpins or primer-dimer formation. I design with Primer3, add 5' sequence cloning sites if necessary (and an overhang), and check hairpins and primer-dimers with the IDT web tool.

i vote for

small Tm diferences between primers (e.g. 1-2°C max.)
no nucleatide repeats (e.g. ...AAA..) more then 4. G repeats are the worse
small primer-primer complementarity
small self-complementarity

my primers must fit all these parameters. Failure = rejection.

P.S: I do not scrutinise my primers for hours on end for complementarity... I use a programe to do that for me

Hi phage434,

With IDT you mean this web tool: http://www.idtdna.com/analyzer/Application...er/Default.aspx?

It looks good. Just did a few tests and the only thing that worries me is that the Tm predictions are 4-5°C smaller than those predicted by primer3. Strange! They both use thermodynamic model. The only difference I could find is an additional K+ term in the IDT equation. Which one do you use for Tm prediction? Primer3 or IDT?

You use a single G or C clamp? Where's your cut off for dimerisation/hairpins?

Thanks for the reply, j

The default settings for Primer3 work for me with an annealing temperature of 55C almost always. I don't look at them much, and certainly don't worry about them. The IDT web site is indeed the one I use. The most important thing is avoiding tight binding of the 3' end of either primer to a location which can extend accidentally. This usually requires 5+ bases matching at the 3' end of the primer. Energy above about 12 Kcal/mole starts to be of concern.

Hi phage434,

So you aim for 55°C. The default settings or primer3 is Min: 57, Opt: 60, Max: 63 (http://frodo.wi.mit.edu/primer3/input.htm). You lower these values to give you primers with Tms around 55? That is working better for you than Tms around 60?

Ta. And all the best,

j

I use the default settings, and then run the PCR reaction with the annealing temperature at 55C.