Oligonucleotide Properties Calculator (Modified by cail)

Enter Oligonucleotide Sequence Below
OD and Molecular Weight calculations are for single-stranded DNA or RNA

Reverse Complement Strand(5' to 3') is:

Reverse Strand(5' to 3') is:

Complement Strand(5' to 3') is:
Number of Fluorescent tags per strand:
6-FAM  TET   HEX   TAMRA  
Minimum base pairs required for single primer self-dimerization:
Minimum base pairs required for a hairpin :
Physical Constants
Melting Temperature (TM)
Length: bases
GC content: %
Molecular Weight: 4
1 ml of a sol'n with an Absorbance of at 260 nm
is microMolar 5 and contains micrograms.
 1 °C (Basic)
2 °C (Salt Adjusted)
3 °C (Nearest Neighbor)
nM Primer
mM Salt (Na+)
Thermodynamic Constants Conditions: 1 M NaCl at 25°C at pH 7.
RlnK cal/(°K*mol) deltaH Kcal/mol
deltaG Kcal/mol deltaS cal/(°K*mol)
Copyright Northwestern University & cail, 1997-2004.

About the Calculations

Thermodynamic Calculations

The nearest neighbor and thermodynamic calculations are done essentially as described by Breslauer et al., (1986) Proc. Nat. Acad. Sci. 83:3746-50 (Abstract) but using the values published by Sugimoto et al., (1996) Nucl. Acids Res. 24:4501-4505 (Abstract). RNA thermodynamic properties were taken from Xia T., SantaLucia J., Burkard M.E., Kierzek R., Schroeder S.J., Jiao X., Cox C., Turner D.H. (1998) Biochemistry 37:14719-14735 (Abstract). This program assumes that the sequences are not symmetric and contain at least one G or C. The minimum length for the query sequence is 8.

Basic Melting Temperature (Tm) Calculations

The two standard approximation calculations are used. For sequences less than 14 nucleotides the formula is

For sequences longer than 13 nucleotides, the equation used is

ASSUMPTIONS:
Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na+, and pH 7.0.

Salt Adjusted Melting Temperature (Tm) Calculations

A variation on two standard approximation calculations are used. For sequences less than 14 nucleotides the same formula as the basic calculation is use, with a salt concentration adjustment

The term 16.6*log10([Na+]) adjusts the Tm for changes in the salt concentration, and the term log10(0.050) adjusts for the salt adjustment at 50 mM Na+. Other monovalent and divalent salts will have an effect on the Tm of the oligonucleotide, but sodium ions are much more effective at forming salt bridges between DNA strands and therefore have the greatest effect in stabilizing double-stranded DNA.
For sequences longer than 13 nucleotides, the equation used is

For oligos longer than 50, the following equation may be more accurate. However, for oligos in the 18-25mer range the above equation (which is what OligoCalc uses) is more accurate.

This equation is most accurate for sequences longer than 50 nucleotides. It is valid for oligos longer than 50 nucleotides from pH 5 to 9. Symbols and salt adjustment term as above, with the term (41 * (yG + zC-16.4)/(wA + xT + yG + zC)) adjusting for G/C content and the term (500/(wA + xT + yG + zC)) adjusting for the length of the sequence, and F is the percent concentration of formamide.

For more information please see the reference:
Howley, P.M; Israel, M.F.; Law, M-F.; and M.A. Martin "A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes." J. Biol. Chem. 254, 4876-4883, 1979.

RNA melting temperatures

Where yG+zC are the mole fractions of G and C in the oligo, L is the length of the shortest strand in the duplex.
ASSUMPTIONS:
These equations assume that the annealing occurs under the standard conditions of 50 nM primer and pH 7.0.

Molecular Weight Calculations

DNA Molecular Weight (for instance synthesized Oligonucleotides)

Anhydrous Molecular Weight = (An x 313.21) + (Tn x 304.2) + (Cn x 289.18) + (Gn x 329.21) - 61.96

An, Tn, Cn, and Gn are the number of each respective nucleotide within the polynucleotide. The subtraction of 61.96 gm/mole from the oligonucleotide molecular weight takes into account the removal of HPO2 (63.98) and the addition of two hydrogens (2.02).

Please note: this calculation works well for synthesized oligonucleotides. If you would like an accurate MW for restriction enzyme cut DNA, please use:

Molecular Weight = (An x 313.21) + (Tn x 304.2) + (Cn x 289.18) + (Gn x 329.21) + 79.0

The addition of 79.0 gm/mole to the oligonucleotide molecular weight takes into account the 5' monophosphate left by most restriction enzymes. No phosphate is present at the 5' end of strands made by primer extension, so no adjustment should be necessary.

RNA Molecular Weight (for instance from an RNA transcript)
Molecular Weight = (An x 329.21) + (Un x 306.17) + (Cn x 305.18) + (Gn x 345.21) + 159.0
An, Un, Cn, and Gn are the number of each respective nucleotide within the polynucleotide. Addition of 159.0 gm/mole to the molecular weight takes into account the 5' triphosphate.

OD Calculations

Molar Absorptivity values in 1/(Moles cm)

ResidueMoles-1 cm-1Amax(nm)Molecular Weight
(after protecting groups are removed)
Adenine (dAMP, Na salt) 15200259313.21
Guanine (dGMP, Na salt) 12010253329.21
Cytosine (dCMP, Na salt) 7050271289.18
Thymidine (dTMP, Na salt) 8400267304.2
RNA nucleotides
Adenine (AMP, Na salt) 15400259329.21
Guanine (GMP, Na salt) 13700253345.21
Cytosine (CMP, Na salt) 9000271305.18
Uradine (UMP, Na salt) 10000262306.2
Other nucleotides
6' FAM 20960 537.46
TET 16255 675.24
HEX 31580 744.13
TAMRA 31980

Assume 1 OD of a standard 1ml solution, measured in a cuvette with a 1 cm pathlength.

6-FAM:

Chemical name: 6-carboxyfluorescein
Absorption wavelength maximum: 495 nm
Emission wavelength maximum: 521 nm
Molar Absorptivity at 260nm: 20960 Moles-1 cm-1

TET:

Chemical name: 4, 7, 2', 7'-Tetrachloro-6-carboxyfluorescein
Absorption wavelength maximum: 519 nm
Emission wavelength maximum: 539 nm
Molar Absorptivity at 260nm: 16255 Moles-1 cm-1

HEX:

Chemical name: 4, 7, 2', 4', 5', 7'-Hexachloro-6-carboxyfluorescein
Absorption wavelength maximum: 537 nm
Emission wavelength maximum: 556 nm
Molar Absorptivity at 260nm: 31580 Moles-1 cm-1

TAMRA:

Chemical name: N, N, N', N'-tetramethyl-6-carboxyrhodamine
Absorption wavelength maximum: 555 nm
Emission wavelength maximum: 580 nm
Molar Absorptivity at 260nm: 31980 Moles-1 cm-1

Nucleotide base codes (IUPAC)

Symbol: nucleotide(s)
    A adenine
    C cytosine
    G guanine
    T thymine in DNA;
    uracil in RNA
    N A or C or G or T
M A or C
R A or G
W A or T
S C or G
Y C or T
K G or T
V A or C or G; not T
H A or C or T; not G
D A or G or T; not C
B C or G or T; not A