Quantitation of Oligonucleotides
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PRIMARY QUANTITATION METHOD (RECOMMENDED)
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UV absorbance at 260 nm (A260 / “OD260”) is a standard method for quantifying nucleic
acids and is accurate when the sample is clean and the correct extinction coefficient is used.
BEST PRACTICES
• Measure ALL samples in duplicate (two independent dilutions is even better).
• Use the AVERAGE of replicate measurements.
• Always record dilution factor and pathlength (1 cm equivalent if using microvolume instruments).
IMPORTANT CLARIFICATION (COMMON CONFUSION)
• For single-stranded DNA (ssDNA):
A260 = 1.0 at 1 cm pathlength corresponds to ~33 µg/mL ssDNA
(this is NOT 33 mg).
• This 33 µg/mL factor is a rough approximation and can be less accurate for short oligos.
MOST ACCURATE UV METHOD FOR OLIGOS
• Use the SEQUENCE-SPECIFIC extinction coefficient (ε260) for the oligo when calculating concentration.
This is more accurate than using a generic “33 µg/mL per A260” conversion.
WHY UV CAN BE WRONG
A260 measures ANYTHING that absorbs at 260 nm, including contaminants such as:
• unincorporated nucleotides
• residual protecting groups or synthesis byproducts
These can inflate A260 and overestimate true oligo mass.
RECOMMENDED CLEANUP BEFORE UV QUANTITATION (WHEN NEEDED)
• Desalt and perform butanol extraction (or equivalent cleanup) prior to A260 quantitation.
• After quantitation, lyophilize if dry material is needed for storage or downstream workflows.
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WHEN FLUOROMETRY IS “MORE ACCURATE” IN PRACTICE
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If samples may contain UV-absorbing contaminants, a fluorometric assay (e.g., ssDNA/oligo
dye-based quantitation such as Qubit ssDNA) is often MORE RELIABLE than UV for concentration.
Use fluorometry when:
• purity is uncertain
• samples may contain free nucleotides/protecting groups
• you need accurate concentration rather than “A260 total absorbance”
Note: fluorometry still measures total nucleic acid present (not “full-length product only”).
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ALTERNATIVE (SEMI-QUANTITATIVE) METHODS
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Polyacrylamide gel estimation
• DNA mass/concentration can be estimated from acrylamide gels.
• Best results: denaturing urea-PAGE (sequencing-style gels).
Visualization: TLC back-shadowing (recommended for gel-based estimation)
• Place gel on a TLC plate and illuminate with a handheld UV source.
• The TLC plate fluoresces; DNA bands absorb UV and appear as dark “shadows.”
• Use as a rough estimate / confirmation, not a primary quantitation method.
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METHODS NOT RECOMMENDED FOR ssDNA/SHORT OLIGOS
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Ethidium staining with transmission fluorescence (typical gel doc)
• Not reliable for quantitating single-stranded oligos.
• Ethidium intercalation depends on double-stranded character.
• Sequence-dependent hairpins cause variable staining intensity.
Agarose gels
• Not reliable for quantitating short, single-stranded oligonucleotides.
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IMPORTANT NOTE ABOUT “FULL-LENGTH PRODUCT” (FLP)
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UV and fluorometry quantify TOTAL nucleic acid, not specifically full-length product.
If you need FLP amount, combine total quantity with a purity estimate (e.g., HPLC/LC peak
area purity) or quantify FLP by an appropriate analytical method.
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SUMMARY (WHAT TO DO)
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1) If sample is clean → UV A260 + sequence-specific ε260 (best UV accuracy)
2) If purity is uncertain → fluorometric ssDNA/oligo assay is often more reliable
3) Use gels for confirmation / rough estimation only
4) Avoid ethidium/agarose for ssDNA oligo quantitation
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