Calculations
Molecular Weight (MW):
The molecular weight of a substance is the weight of one mole of a that
substance. To calculate the molecular weight of an oligonucleotide, the molecular
weights of the individual nucleotides present in the oligo must first be summed.
Then the weight must be corrected by subtracting 62.97, which is equal to two oxygen
atoms and a phosphorus atom, to account for the 5’-terminal OH group, and
by adding 1.008, which is equal to one hydrogen atom, to account for the 3’-terminal
OH group.
The nucleotide unit:
For DNA: R = H; “Base” can be Adenine, Cytosine, Guanine, or Thymine.
For RNA R = OH; “Base” can be Adenine, Cytosine, Guanine, or Uracil.
DNA: MW = (#dA x 314.21) + (#dC x 289.18) + (#dG x 329.21) + (#dT x 304.19) –
62.97 + 1.008
RNA: MW = (#A x 330.21) + (#C x 305.18) + (#G x 345.21) + (#U x 306.17) –
62.97 + 1.008
To calculate the molecular weight of Oligo Potassium Salts, the molecular weight
of the normal oligonucleotide must first be calculated (see above). This weight
is then corrected by adding 39.1, which is equal to one potassium atom, and subtracting
1.008, which is equal to one hydrogen atom. This correction accounts for replacing
all the acidic protons on the backbone with potassium ions.
K-Salt: MW = MW(normal) + (# nucleotides -1) x (39.1 - 1.008)
Absorbance (Optical Density)
The concentration of oligonucleotide in a sample is measured by its absorbance at
260 nm according to the Beer-Lambert law.
where A is the absorbance (optical density or OD), ε is the molar extinction coefficient,
c is the concentration, and l is the pathlength.
Extinction Coefficient (e260):
e260,
the extinction coefficient at 260nm, is a constant that indicates the extent to
which a given DNA or RNA strand in solution absorbs light at 260nm.
In general, the calculation of the extinction coefficient of an oligonucleotide
of length N can be given by the expression:
eN is the “nearest-neighbor”
extinction coefficient of neighboring nucleotides i and i +
1.
Nearest-neighbor extinction coefficients at 260nm:
|
|
i = C
|
i = G
|
i = A
|
i = U
|
i = dC
|
i = dG
|
i = dA
|
i = dT
|
|
i + 1 = C
|
14200
|
17800
|
21000
|
16200
|
14400
|
17900
|
21050
|
15200
|
|
i + 1 = G
|
17400
|
21600
|
25200
|
21200
|
17500
|
21600
|
25200
|
20000
|
|
i + 1 = A
|
21000
|
25000
|
27400
|
24000
|
21100
|
25000
|
27400
|
22800
|
|
i + 1 = U
|
17200
|
20000
|
24600
|
19600
|
17200
|
20000
|
24600
|
18200
|
|
i + 1 = dC
|
14400
|
17900
|
21050
|
16200
|
14600
|
18000
|
21100
|
15200
|
|
i + 1 = dG
|
175000
|
21600
|
25200
|
21200
|
17600
|
21600
|
25200
|
20000
|
|
i + 1 = dA
|
21100
|
25000
|
27400
|
24000
|
21200
|
25000
|
27400
|
22800
|
|
i + 1 = dT
|
16200
|
19000
|
23400
|
18200
|
16200
|
19000
|
23400
|
16800
|
eI< is the “individual”
extinction coefficient for nucleotide i.
Individual extinction coefficients at 260nm:
|
i = C
|
i = G
|
i = A
|
i = U
|
i = dC
|
i = dG
|
i = dA
|
i = dT
|
|
7200
|
11500
|
15400
|
9900
|
7400
|
11500
|
15400
|
8700
|
Picomoles (pmol):
Picomole is a measurement of amount. The following equation can
be used to calculate picomoles from the optical density (OD) and extinction coefficient
(e260) of the oligonucleotide.
Micrograms (µg):
Microgram is a measurement of weight. The following equation can
be used to calculate micrograms from the picomoles (pmol) and molecular weight (MW)
of the oligonucleotide.
Note: for an oligonucleotide 1 OD260 = 33µg.
Molarity (M):
Molarity is a measurement of concentration. It is equivalent to moles (mol)
of solute per liter (L) of solution as expressed in the equation below.
Melting Temperature (Tm):
The melting temperature of an oligonucleotide is dependent upon
a number of factors including the length and G+C content of the sequence as well
as the type and concentrations of cations present, particularly the sodium ion,
Na+. A variety of formulas have been used for predicting melting temperatures. The
following formula is recommended for oligonucleotides ranging in length from 20
to 100 residues and sodium concentrations ([Na+]) ranging from 0.01 M to 1.0 M.
Annealing Temperature for PCR:
To calculate the annealing temperature of oligonucleotides, first
calculate the melting temperature (see above), then subtract approximately 5 - 10
degrees. If the two oligonucleotides have different melting temperatures do NOT
average the numbers. Use the lower number so that both of the oligonucleotides can
anneal. For 10mers, Eurofins MWG Operon recommends that PCR be done at 32-33°C
in order to maximize repeatability.
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