For Primers Less Than 14 Bases in Length

• 1). Add the number of guanines and cytosines in your primer sequence, and then add up the number of adenines and thymines.
  
  
• 2). Multiply the total guanines plus cytosines by four, and multiply the total adenines plus thymines by two.
  
  
• 3). Add the two totals together for melting temperature (Tm) of your primers, and then subtract five for optimal Ta. Thus, Ta = [4(GC) + 2(AT)]-5.
  
  

For Primers Greater Than 14 Bases in Length

• 1). Find the total number of guanines plus cytosines in your primer.
  
  
• 2). Plug the total guanines plus cytosines into the formula provided by Promega: "Tm = 64.9 degrees C + 41 degrees C x (number of G's and C's in the primer -- 16.4)/N where N is the length of the primer."
  
  
• 3). Subtract five from this total for Ta. Thus, Ta = [64.9 degrees C + 41 degrees C(total G+C -- 16.4)/N] -- 5.
  
  
• 4). Use the simplest formula instead: Ta = 3(total guanines plus cytosines) + 2(total adenines plus thymines) .