Calculating Oligonucleotide Quantity
CALCULATIONS & CONVERSION FACTORS
SPECIFICATIONS SHEET — CALCULATIONS & CONVERSION FACTORS
PURPOSE
Use these formulas and conversion factors to calculate the quantity and molecular weight
of synthetic oligonucleotides.
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OD260 MEASUREMENT
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• The absorbance at 260 nm (OD260) is measured at TWO different dilutions.
• The OD260 value reported on the specification sheet is the AVERAGE of these two readings.
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QUANTITY CALCULATIONS (FROM OD260)
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CONVERSION FACTOR (SINGLE-STRANDED DNA)
• 1 OD260 = 33 µg = 0.033 mg
CALCULATIONS
1) Milligrams (mg)
mg = OD260 × 0.033
2) Nanomoles (nmol)
nmol = (mg ÷ molecular weight) × 10^6
NOTE
• The molecular weight (MW) for each oligonucleotide is listed on that oligo’s specification sheet.
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MOLECULAR WEIGHT CALCULATION
(SYNTHETIC OLIGONUCLEOTIDES)
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FORMULA
Molecular Weight (g/mol) =
(251 × nA) +
(245 × nT) +
(267 × nG) +
(230 × nC) +
(61 × (n − 1)) +
(54 × n) +
(17 × (n − 1)) +
2
WHERE
• nA = number of dA bases
• nT = number of T bases
• nG = number of dG bases
• nC = number of dC bases
• n = total number of bases in the sequence
TERM DEFINITIONS
• (61 × (n − 1)) accounts for the molecular weight of the phosphate groups.
• (54 × n) accounts for hydration of the DNA (≈ 3 water molecules per nucleotide).
• (17 × (n − 1)) accounts for ammonium cations associated with the phosphate groups.
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SAMPLE CALCULATION
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Example sequence:
ATG TAA TGT TTG GTC CGC
Base counts:
• nA = 3
• nT = 7
• nG = 5
• nC = 3
• n = 18
Calculated molecular weight:
• 6,793 g/mol
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MODIFICATIONS
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Special modifications (e.g., biotin, 5′ phosphate, inosine, uridine, etc.)
add additional mass to the oligonucleotide and must be included separately
when calculating molecular weight.