Primer tm - (May/22/2009 )
Hello all, I am trying to amplify a 5kb gene. There's also this similar-gene which is about 3 kb, so I have used Primer3 to pick primers excluding the matching regions of the two. F-GTG AGG GGA GGC GGT TGT AG and Reverse GTG ACG GAA AAT GCT TAC TTA CCC. When i used primerstat to find the melting temp, the three algorithms give values a li'l far apart- Basic Tm (degrees C): 58, Salt adjusted Tm (degrees C): 53 and Nearest neighbor Tm (degrees C): 67.26 for the first and Basic Tm (degrees C): 56 Salt adjusted Tm (degrees C): 51
Nearest neighbor Tm (degrees C): 64.70 for the reverse. Do I have to redesign the primers, or can I make do with these?
2 degrees difference in melting temperature shouldn't be a problem.
If you have already ordered them: Just try it out!
If you haven't ordered them yet: add/delete about 2 nucleotides from one of the primers to get the same Tm...