Looking for an annealing temperature calculator that uses the Wallace et al equation to calculate NEB TM online? Then you've come to the right place!
Annealing temperature (Tm) is the temperature at which primers or primers bind or “anneal” (form hydrogen bonds) to complementary strands of DNA, allowing extension by the polymerase. The optimal annealing temperature depends on primer sequences and reaction conditions such as reaction buffer, ion concentration, and primer concentration. The correct choice of annealing temperature is crucial to obtain accurate and specific results in PCR reactions.
There are several equations available to calculate the PCR annealing temperature, the most commonly used equation by Wallace et al. This equation takes into account primer sequences and primer concentration, as well as the number of GC base pairs in the primer sequence. The formula for calculating Tm is as follows:
Tm = 64.9 + 41*(G + C - 16.4)/(A + T + G + C - 16.4 - 2*P) - 500/(A + T + G + C + 0.41*P)
Where:
Tm = Annealing temperature in degrees Celsius A, T, G, C = Number of adenine, thymine, guanine and cytosine nucleotides, respectively, in the primer sequence P = Number of GC base pairs in the primer sequence
Wallace et al. is just one of several equations available to calculate Tm, but it is widely used because of its accuracy and ease of use.
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Ring Temperature Calculator Wallace et al Equation
The calculator uses the Wallace et al. to calculate Tm based on primer sequences and primer concentration.
About our NEB TM Calculator
The calculator is designed for use with NEB PCR products. The first step is to select the NEB PCR product that you plan to use in the PCR reaction. This will help determine the appropriate reaction conditions and the correct formula for calculating the Tm.
To use, enter the primer sequences into the calculator. Be sure to enter the sequences in the correct order - first starter in the first box and second starter in the second box. The calculator allows up to 3 ambiguous bases (represented by the codes "N", "R", "Y", etc.) in the primer sequences.
After entering the required information, click the "Calculate Tm" button. The calculator will use the Wallace et al. to calculate Tm based on primer sequences and primer concentration.
The calculator will display the calculated Tm in degrees Celsius in the text box below the "Calculate Tm" button. Make sure the calculated Tm is within the proper temperature range for the NEB PCR product you are using.
Other Ways to Calculate Ring Temperature
In addition to the Wallace et al. equation, there are other equations that can be used to calculate the PCR annealing temperature based on primer sequences. Here are some other common equations:
Breslauer et al formula
The formula by Breslauer et al. is one of the oldest and most common equations for calculating PCR annealing temperature. The equation is given by:
Tm = 81.5 + 0.41*(G + C) - 675/N - P + S
Where:
Tm = Annealing temperature in degrees Celsius G, C = Number of guanine and cytosine nucleotides, respectively, in the primer sequence N = Total number of nucleotides in the primer sequence P = Number of base pairs in the primer sequence S = Salt concentration ( Na+) in mol/L
This equation is relatively simple, but it may not be as accurate as other more recent equations.
Formula by SantaLucia et al.
The formula by SantaLucia et al. is a newer and more accurate equation for calculating the PCR annealing temperature. The equation is given by:
Tm = 100.5 + 41*(G - C/2)/(A + T + G + C) - 820/dH - 16.6*log10(S/1000)
Where:
Annealing temperature in degrees Celsius G, C = Number of guanine and cytosine nucleotides, respectively, in the sequence of the primers A, T = Number of adenine and thymine nucleotides, respectively, in the sequence of the primers dH = Enthalpy of denaturation of the primer in cal/mol S = Salt concentration (Na+) in mol/L
This equation takes into account the enthalpy of denaturation of the primers, which can be measured experimentally, as well as the salt concentration in the PCR reaction.
