is anybody working on gibson assembly commercialized by NEB? NEBuilder software also helps in designing primer etc. My question is, software predicted annealing temperature differs significantly for taq polymerase and fusion polymerase. Why is it?
NEBuilder calculates annealing temperatures for the Q5 HF Polymerase. You can change the polymerase in "Set preferences", --> "Change Prefs".
Best regards!
@Juergen is right. It is important to set the proper polymerase. In addition, the underlying thermodynamic parameters used for Phusion Tm calculations are different from all the other polymerases. It uses modified Breslauer data, as originally recommended by Invitrogen, whereas all other calculations use data from SantaLucia. This difference was required to maintain compatibility with the original Tm estimates provided by the old Invitrogen (now Thermo Fisher Scientific) Phusion Tm calculator. Hope that clears up the reason for the significantly different Tm values.
It predicts different Tm because buffer composition (such salt concentration, presence of detergents..) for those two enzymes is different. The calculator take into account this info.
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