primer design: GC content v/s amplicon structure

[scuellar]

Hi every one,
When designing qPCR primers, what is better: a high GC content in the primers (I mean 65 to 70%) including things like ccg or ggg but low amplicon structure at 60C (I mean dG around 0.6 kcal/mol), or GC content lower than 60%, and amplicon structure dG between 2 and 3 kcal/mol?

I know at the end every primer pair should be tested but if I can choose the best designed ones will spend less time and money in the optimization, ideas from the experience?

Thanks

[pc88]

Hi everyone! I have read the MIQE guidelines and the article refers the importance to assess the annealing of primers with suitable RNA regions (e.g: without hairpins). My question is if there is a good software to overlap primer sequences with RNA secondary structure? Moreover, I though that secondary structures were denaturated with the high temperature cycles... if that's the case... it doesn´t make sense my previous question xD

Can anybody help?

thanks

[namishu95]

Hi every one,
When designing qPCR primers, what is better: a high GC content in the primers (I mean 65 to 70%) including things like ccg or ggg but low amplicon structure at 60C (I mean dG around 0.6 kcal/mol), or GC content lower than 60%, and amplicon structure dG between 2 and 3 kcal/mol?

I know at the end every primer pair should be tested but if I can choose the best designed ones will spend less time and money in the optimization, ideas from the experience?

Thanks

[namishu95]

the importantance for probe design is to have Tm of probe as 6-8 degree higher than primer, i usually design probe to have Tm as 66-68 degree, and it is better to have GC content as 40-60%,