--- title: How to Analyze and QC Oligonucleotide Samples? description: Recommended QC techniques for oligos --- [Skip to content](https://support.sierrabio.com/how-to-analyze-and-qc-oligonucleotide-samples#main-content) English Show submenu for translations ![](https://support.sierrabio.com/hs-fs/hubfs/JPG%20file%20-02.jpg?width=2000&height=1125&name=JPG%20file%20-02.jpg) Open main navigation Close main navigation - English Show submenu for translations - [Contact us](mailto:support@sierrabio.com) [Contact us](mailto:support@sierrabio.com) How can we help you? - There are no suggestions because the search field is empty. 1. [Sierra Biosystems Support Center](https://support.sierrabio.com/?hsLang=en) 2. [Oligonucleotide Chemistry & Methods](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en) 3. [Quantitation & QC](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en#quantitation-qc) September 19, 2025 # How to Analyze and QC Oligonucleotide Samples? ## Ensuring the purity, concentration, and structural integrity of synthetic oligonucleotides (oligos) is critical for successful downstream applications like PCR, qPCR, sequencing, or therapeutic research. This article outlines the most widely used analytical and QC methods, their strengths, and when to use them. #### 1. UV-Vis Spectroscopy (Nanodrop or Spectrophotometer) **What it tells you:** - Concentration of oligo (OD 260) - Approximate purity **How it works:** Oligos absorb UV light at 260 nm. By measuring absorbance, you can calculate concentration using Beer–Lambert law. Most labs use Nanodrop or quartz cuvettes for this quick check. **Example workflow:** 1. Dissolve your dried oligo in sterile water or buffer. 2. Dilute a small aliquot if absorbance is above linear range. 3. Measure at 260 nm. 4. Use the extinction coefficient (sequence-dependent) to calculate nmol or µg. **Rules of thumb:** - 1 OD ≈ 5 nmol for a 20mer DNA oligo. - 1 OD ≈ 30 µg DNA oligo. **Limitations:** - Cannot distinguish between full-length product and truncated/impure sequences. --- #### 2. High-Performance Liquid Chromatography (HPLC) **What it tells you:** - Separation of full-length vs truncated oligos - Relative purity **How it works:** HPLC separates molecules based on polarity or size. Reverse-phase or ion-exchange HPLC is most common for DNA/RNA QC. **When to use it:** - Assessing purity after synthesis - Removing short fragments or impurities - Preparative purification **Strengths:** - Good resolution - Effective for separating truncated products - Can be scaled from analytical to preparative runs **Limitations:** - Does not provide molecular weight confirmation. --- #### 3. Mass Spectrometry (MS) **What it tells you:** - Exact molecular weight - Presence of modifications - Identity of impurities **How it works:** Coupling LC to MS enables both quantification (UV trace) and **mass confirmation**. Even truncated sequences (N-1), depurination products, or chemical adducts can be detected. **Key applications:** - Confirming oligo mass (ensuring the right sequence was synthesized) - Detecting impurities at \<5–10% levels (e.g., depurination, protecting group remnants) - Characterizing modifications **Strengths:** - Gold standard for identity confirmation - High sensitivity for impurities **Limitations:** - Requires specialized equipment and expertise - Ion-pairing reagents may complicate analysis if not managed properly --- #### 4. PAGE (Polyacrylamide Gel Electrophoresis) **What it tells you:** - Oligo size distribution - Presence of truncated products **How it works:** Oligos migrate based on charge and size in a polyacrylamide gel. Bands can be visualized with stains or radiolabels. **When to use it:** - Useful for long oligos (\>50 bases) - Comparing integrity of multiple samples - Educational or troubleshooting tool **Strengths:** - High resolution separation by length - Inexpensive and accessible **Limitations:** - Semi-quantitative at best - Labor-intensive compared to HPLC/MS --- #### Putting It All Together: A QC Strategy No single method answers all QC questions. The most effective approach is **layered analysis**: | QC Goal | Best Method(s) | | --- | --- | | Quick concentration check | UV-Vis / Nanodrop | | Purity assessment | HPLC + UV detection | | Confirm correct sequence | Mass spectrometry | | Size distribution (long oligos) | PAGE | | Impurity identification | LC-MS | **Recommended workflow:** 1. **Start with UV** → concentration check. 2. **Run HPLC** → get a purity profile. 3. **Confirm with MS** → verify mass and check for impurities. 4. **Add PAGE if needed** → especially for long RNA/DNA strands. --- #### Quick Reference Equations - **Concentration (Beer–Lambert Law):** *A = ε × C × l* (where A = absorbance, ε = extinction coefficient, C = concentration, l = path length) - **Molecular Weight of Oligo:** MW = (313.2 × A) + (329.2 × G) + (289.2 × C) + (304.2 × T) – 61 ± modifications --- ✅ **Tip:** For high-value or modified oligos (e.g., siRNA, probes), always perform MS confirmation before use in experiments.   - [Pre-Installation & Installation](https://support.sierrabio.com/pre-installation-installation?hsLang=en#main-content) - [Pre-Installation Requirements](https://support.sierrabio.com/pre-installation-installation?hsLang=en#pre-installation-requirements) - [Site & Lab Preparation](https://support.sierrabio.com/pre-installation-installation?hsLang=en#site-lab-preparation) - [Installation & Initial Setup](https://support.sierrabio.com/pre-installation-installation?hsLang=en#installation-initial-setup) - [Troubleshooting & Errors](https://support.sierrabio.com/troubleshooting-errors?hsLang=en#main-content) - [Software Errors & Communication Issues](https://support.sierrabio.com/troubleshooting-errors?hsLang=en#software-errors-communication-issues) - [Chemistry and Protocol-Related Issues](https://support.sierrabio.com/troubleshooting-errors?hsLang=en#chemistry-and-protocol-related-issues) - [K&A - Preventative Maintenance & Quick Fixes](https://support.sierrabio.com/troubleshooting-errors?hsLang=en#ka-preventative-maintenance-quick-fixes) - [Shasta - Preventative Maintenance & Quick Fixes](https://support.sierrabio.com/troubleshooting-errors?hsLang=en#shasta-preventative-maintenance-quick-fixes) - [Shasta - Hardware Issues](https://support.sierrabio.com/troubleshooting-errors?hsLang=en#shasta-hardware-issues) - [Training Videos and Guides](https://support.sierrabio.com/training-videos-and-guides?hsLang=en#main-content) - [Shasta Software](https://support.sierrabio.com/training-videos-and-guides?hsLang=en#shasta-software) - [K&A Next Gen Software](https://support.sierrabio.com/training-videos-and-guides?hsLang=en#ka-next-gen-software) - [K&A Legacy Software](https://support.sierrabio.com/training-videos-and-guides?hsLang=en#ka-legacy-software) - [K&A - Troubleshooting Videos](https://support.sierrabio.com/training-videos-and-guides?hsLang=en#ka-troubleshooting-videos) - [Shasta - Troubleshooting Videos](https://support.sierrabio.com/training-videos-and-guides?hsLang=en#shasta-troubleshooting-videos) - [Software Updates & Release Notes](https://support.sierrabio.com/software-updates-release-notes?hsLang=en#main-content) - [Release Notes](https://support.sierrabio.com/software-updates-release-notes?hsLang=en#release-notes) - [Downloads](https://support.sierrabio.com/software-updates-release-notes?hsLang=en#downloads) - [Update Instructions](https://support.sierrabio.com/software-updates-release-notes?hsLang=en#update-instructions) - [Product Documentation & Policies](https://support.sierrabio.com/product-documentation-policies?hsLang=en#main-content) - [Technical Documentation](https://support.sierrabio.com/product-documentation-policies?hsLang=en#technical-documentation) - [Specifications](https://support.sierrabio.com/product-documentation-policies?hsLang=en#specifications) - [Warranty & Service](https://support.sierrabio.com/product-documentation-policies?hsLang=en#warranty-service) - [Oligonucleotide Chemistry & Methods](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en#main-content) - [Synthesis & Strategy](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en#synthesis-strategy) - [Post-Synthesis Processing](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en#post-synthesis-processing) - [Quantitation & QC](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en#quantitation-qc) - [Special Topics](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en#special-topics) - [Support & Contact](https://support.sierrabio.com/support-contact?hsLang=en) - [Uncategorized](https://support.sierrabio.com/uncategorized?hsLang=en) [![](https://support.sierrabio.com/hs-fs/hubfs/JPG%20file%20-02.jpg?width=2000&height=1125&name=JPG%20file%20-02.jpg)](http://sierrabio.com) [mailto:Support@sierrabio.com](mailto:Support@sierrabio.com) Copyright © 2025, Sierra BioSystems