primer design - how far apart Tms could be? (Apr/10/2008 )
i´ve designed primers with Tm 60 and 64. are they too far apart to run pcr?
I guess it should be ok. Mine was around 62 and 64 degrees and it worked well. Take care to set the right annealing temperature.
I was taught that up to 5 degrees difference is ok.
Within 5° is considered optimal, but I've run primers that were up to 10° apart and still worked. (though that's kind of pushing it)... yours should be fine. Just make sure your annealing temp is at least 5°C below the lowest TM.
thanks to all suggestions.