Can you please tell what is "cisono special conditions"?

excuse me.... are there special condition?

Ciao Emilia, probably such long primers are not all annealing to your template, I can imagine you have a 3' side (maybe 15-20 nts) that is annealing and a 5' tail that you will need for something else. The part that is really annealing will determine the PCR conditions, and normally you don't need to change them even if after some cycles you'll have an increasing amount of template containing all 66 nt of your primers, since the abundance of the specific template build-up will rule out unspecific binding of your primers.

In any case, annealing temperature is not linearly correlated with the DNA length. You are able to fully denature million nt long genomic DNA at 96°C, therefore annealing temperature changes significantly the shorter the primers, but much less the longer they are. Therefore, even if your 66 nt long primers are all annealing to the template you might want to work between 62 and 65°C for the annealing. At worst, you might try a two step PCR cycling at 96 and 68°C only.

Finally, keep in mind that your template will strongly determine your results. Genomic DNA is pretty difficult, and using long primers will increase the chance of their unspecific binding, titrating them away from your specific reaction, which means getting your amplicon might be very hard work. mRNA is much easier to amplify, but if you will have a plasmid template, then practically any condition will easily give you the amplified band.

I do not really understand the word "CISONO"

I have been using 80nt long primers for introducing regions onto the PCR product.

As such there are no special conditions. We use the regular 1pico molar conc. of the primers in as much PCR reaction that your manufacturer suggest like with PlatinumTaq HIFI 1ul of 1pmol primer for 50ul reaction. The quantity varies from one enzyme to other but generally 1ul for 25ul reaction works mostly. 

The only concern is that you cant control the aneeling temp as long primers have high Tm. Thus you give it the highest aneeling temp that you can give but still it would be appreciably low than the Tm, Thus you would end up with non-specific products along with your intended product and you will have to gel purify your product. 

 no special conditions, I have been using 50nt long primers for amplification.

maybe my question have not been very clear: I would like to amplify a small viral region of 189nt, using a pair of primers shorter and a second pair of primers of 66 nt in the same pcr reaction and i would know if is need to use different concentrations for each primers...so, i will be performed a pcr with different Ta in the same reaction.. thanks everyone for your explanations

I dont think special conditions are required to amplify using longmers. Just use 72c annealing cum extension temperature for product specific time period. You mentioned you are trying to amplify 189 bps target. So you should be giving 25 to 30 sec at 72c. This should be enough. Using longer time periods could yield non-specific products. If you have seen significant non-specific product generation, then lower the divalent cation concentration and you can be sure to have reduced non-specificity. This will lower prodcut yield too. 

If you are using external primers to amplify the product generated by the internal primers, then use lower internal primer concentration and high external primer conc. say 1:5 is to be used. 

I think this might be an issue as if the difference of Tm is significant you will end up with non specific products. If possible go for two step PCR approach otherwise I think one idea might be to use high aneeling temp which is right for long primer and at the same time increase the short primer conc. I have not played much with it. But once technician made the mistake of using 60 as aneeling temp where the Tm for pair was 57 and she said she would get product with twice the normal amount of Primers but not with normal conc. So probably you could play around a little with that. Hope it would help. 

Thanks...i will inform you

You explained your experiment to some extent, but it is not yet clear  to me. So your target of interest is 189 NT and you want to perform a kind of nested PCR in a duplex reaction. Is this a design for conventional or real time PCR?