Use Annealing Temp Rule when you need to estimate the PCR annealing temperature as the lower primer melting temperature minus five degrees. Enter Forward primer Tm and Reverse primer Tm using the definitions and units below so the result can be reproduced.

Forward primer Tm

A finite measured or assumed value, expressed in °C.

Reverse primer Tm

A finite measured or assumed value, expressed in °C.

The published tool contract uses this rule: Estimate the PCR annealing temperature as the lower primer melting temperature minus five degrees.

Annealing temperature

The calculated annealing temperature in °C.

The output is an estimate tied to the stated model and coefficients; it is not a direct measurement and should retain that label when reported.

Retain the original Forward primer Tm and Reverse primer Tm values and their units alongside the result. Nearest Neighbor Tm Approx is useful when you instead need to approximate primer melting temperature with a salt-adjusted nearest-neighbor style formula. GC Rule Tm provides another view by helping you estimate DNA melting temperature with the GC rule for sequences longer than 13 bases.

• Nearest Neighbor Tm Approx

  Approximate primer melting temperature with a salt-adjusted nearest-neighbor style formula.

• GC Rule Tm

  Estimate DNA melting temperature with the GC rule for sequences longer than 13 bases.

• GC Clamp Check

  Check the GC clamp of a primer by counting guanine and cytosine bases among the last five nucleotides.