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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. smile.gif


thanks to all suggestions.