Primer Melting Temperature Calculator

We’ve created this primer melting temperature calculator that measure temperature at which half of the DNA primer-template duplexes dissociate into single strands.

This critical temperature, known as the melting temperature (Tm), is vital for designing effective primers for PCR (Polymerase Chain Reaction) and other DNA amplification techniques.

Consider a primer sequence ATGCCTGACTAG. The calculator evaluates factors such as sequence length, GC content, and salt concentration to estimate the Tm. This information is crucial for researchers to optimize PCR conditions for efficient and specific DNA amplification.

Primer Melting Temperature Calculation Chart

Primer Sequence	Length	GC Content (%)	Salt Conc. (mM)	Estimated Tm (°C)	3′ End Base	Recommended Use
ATGCCTGACTAG	12	50	50	36.0	G	General PCR
GCGCGCGCGCGC	12	100	50	52.0	C	High specificity PCR
ATATATATATAT	12	0	50	20.0	A	Low complexity targets
CGTACGTACGTA	12	50	100	38.0	A	Standard PCR
TGCATGCATGCA	12	50	25	34.0	C	General PCR
GATCGATCGATC	12	60	50	40.0	G	Cloning applications
CCTAGGATCCTAG	12	45	75	37.5	C	qPCR
TTGACCTGACCT	12	50	100	39.0	G	Multiplex PCR
AGCTAGCTAGCT	12	50	50	36.5	C	Genomic DNA amplification
TGACGATCGTAG	12	55	50	41.0	G	High-throughput sequencing

Primer Melting Temperature Formula

A commonly used simple formula for primers shorter than 14 nucleotides is:

Tm = 2°C (A + T) + 4°C (G + C)

Where:

A, T, G, C = number of respective bases

Tm = Melting temperature in °C

For longer primers (14-70 nucleotides), a more accurate formula is:

Tm = 64.9°C + 41°C * (number of G's and C's - 16.4) / Length

Salt-Adjusted Formula:

Tm = 81.5 + 16.6(log10[Na+]) + 0.41(%GC) - 675/N

Where:

[Na+] = molar sodium concentration
%GC = percentage of G and C bases
N = total length of primer

Nearest-Neighbor Method:

Tm = ΔH/(ΔS + R × ln(c/4)) - 273.15 + 16.6 × log10[Na+]

Where:

ΔH = sum of nearest-neighbor enthalpy changes
ΔS = sum of nearest-neighbor entropy changes
R = gas constant (1.987 cal/°C×mol)
c = primer concentration
[Na+] = molar sodium concentration

GC Content Calculation:

%GC = (G + C)/(A + T + G + C) × 100

Correction Factors:

For DMSO: Tm = Tm - 0.6°C per 1% DMSO
For Formamide: Tm = Tm - 0.6°C per 1% formamide

Using the simple formula:

Tm = 2°C (3 + 3) + 4°C (3 + 3)
Tm = 2°C 6 + 4°C 6
Tm = 12°C + 24°C = 36°C

Using the longer primer formula:

Tm = 64.9°C + 41°C * (6 – 16.4) / 12
Tm = 64.9°C – 35.53°C
Tm ≈ 29.37°C

The difference in results highlights the importance of using appropriate formulas and considering additional factors like salt concentration for more accurate predictions.

How to Calculate the Melting Temperature of a Primer?

To calculate the melting temperature of a primer:

Determine the primer sequence and length.
Count the number of each nucleotide (A, T, G, C).
Calculate the GC content (percentage of G and C bases).
Consider the salt concentration of the solution.
Apply an appropriate formula based on the primer length and experimental conditions.

Calculate the Tm for the Primer GCATTAGCGTA in 50mM NaCl

Sequence: GCATTAGCGTA, Length: 11 nucleotides
Nucleotide count: G=3, C=2, A=3, T=3
GC content: (3+2) / 11 ≈ 45.5%
Salt concentration: 50mM NaCl

Using the nearest-neighbor method (a more accurate approach for considering sequence context):

Tm = 81.5 + 16.6 log10([Na+]) + 0.41 (%GC) - 675 / length

Tm = 81.5 + 16.6 log10(0.05) + 0.41 45.5 – 675 / 11
Tm ≈ 35.8°C

This calculated Tm helps researchers determine the optimal annealing temperature for PCR, typically set a few degrees below the Tm to ensure specific primer binding while allowing for efficient extension by the polymerase enzyme.

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Primer Melting Temperature Calculator