---
title: Synthesizing Long Oligos
description: "Long Oligonucleotide Synthesis: Challenges, Best Practices, and Troubleshooting"
---

[Skip to content](https://support.sierrabio.com/synthesizing-long-oligos#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. [Synthesis & Strategy](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en#synthesis-strategy)

September 3, 2026

# Synthesizing Long Oligos

## Long Oligonucleotide Synthesis: Challenges, Best Practices, and Troubleshooting

### Why this matters

Long oligo synthesis is fundamentally less forgiving than short oligo synthesis. As length increases, small inefficiencies compound rapidly, leading to:

- lower full-length yield
- higher deletion/truncation levels
- more variability run-to-run
- more purification difficulty

This article outlines the most important factors that control long-oligo success and how to troubleshoot when results drift. For the specific chemistry and support changes to make at 100 bases and above, see [Long Oligos (100+ Bases): Chemistry and Support Changes](https://support.sierrabio.com/long-oligos-100-bases?hsLang=en).

---

### 1) Key Principle: Small Efficiency Losses Become Big Problems

Every cycle (detritylation, coupling, capping, oxidation) must stay consistent and efficient.  
A small drop in performance early or mid-run can dramatically reduce the final full-length fraction.

**Bottom line:** long-oligo success depends on stable, repeatable performance across *all* cycles.

---

### 2) Steric Hindrance (Additional Steps May Be Needed)

As the oligonucleotide grows in length, steric hindrance increases, which can reduce reagent access and slow reaction completion across the synthesis cycle. To maintain full-length yield and purity on long sequences, it may be necessary to increase reaction time or add additional repetitions of one or more steps, including deblock (detritylation), coupling, capping, and oxidation, especially when performance declines later in the run or long-oligo impurity levels increase.

---

### 3) Support Selection

#### **Why the solid support matters more on long sequences**

As an oligo grows, the support surface becomes crowded. This can slow diffusion of reagents into the support and make reactions less consistent over time.

Common impacts include:

- slower reagent penetration
- reduced coupling uniformity later in the run
- increased truncation and mixed impurities

#### General guidance for long oligos

For longer targets, generally above 100 nt, supports with **larger pore structures** often provide better access for reagents as strand length increases.

**Tradeoffs to consider**  
Supports optimized for long sequences may introduce different practical constraints such as:

- lower loading capacity
- increased fragility
- more sensitivity to handling and column packing

**Standard (Functionalized) vs Universal**

- Standard support includes the first 3’ base, so you get one less coupling step.
- It often allows simpler / less harsh cleavage conditions.
- Universal support is more flexible, but typically requires an extra coupling and can require harsher cleavage to fully release the oligo

**Best practice**

Choose support type and loading level based on what you care about most:

- maximum yield
- maximum purity
- maximum repeatability

For long oligos, **repeatability** is usually the best starting target.

---

### 4) Coupling Step (Highest Impact Chemical Variable)

#### **Why coupling is the primary yield driver**

Coupling is the step where the next base is added to the growing chain. For long oligos, coupling must remain extremely consistent across dozens or hundreds of cycles.

Even small coupling losses are amplified with length.

#### **The #1 coupling risk: moisture (water contamination)**

Moisture lowers coupling efficiency in two main ways:

**A) Water competes with the intended reaction**  
Activated monomer should react with the growing strand. Water can consume activated species instead, lowering coupling yield.

**B) Water gradually reduces monomer effectiveness**  
Moisture exposure over time can degrade monomers while they sit on the instrument, causing coupling to worsen later in the run.

#### **Common causes of moisture exposure**

- humid environments
- solvent bottles not sealed tightly
- older acetonitrile that absorbed moisture
- gas supply not sufficiently dry
- instruments sitting unused (internal plumbing equilibrates to ambient moisture)

#### **Best practices to protect coupling efficiency**

- Use dry acetonitrile (instrument + monomer dilution)
- Use trap packs or molecular sieves to further ensure dryness
- Prepare fresh monomer solutions and minimize open-air exposure
- Keep activator clean, fresh, and properly stored
- Ensure inert gas (argon/helium) is dry and leak-free
- After downtime, consider running a conditioning/purge routine before critical long-oligo runs

---

### 5) Capping Step

#### **What capping is supposed to do**

Capping blocks strands that failed to couple in that cycle, preventing them from reacting in later steps.

#### **Why capping issues hurt long oligos more**

If capping is incomplete:

- failure strands stay reactive
- they continue growing
- they form deletion impurities (n–1 family)

These deletion impurities are especially problematic because they can be very difficult to remove during purification.

#### **Signs of weak capping performance**

- high levels of deletion impurities
- purity that does not improve despite “okay” coupling metrics
- inconsistent final product purity even when yield looks decent

#### **Best practices for capping on long oligos**

- Keep capping conditions consistent across the full run
- If your system allows it, increase:
- - capping volume
    - contact time  
      (especially when long-oligo purity is the priority)
    - Confirm capping reagent integrity and line function (no partial delivery).

---

### Quick Troubleshooting Guide

#### **If full-length yield is low across the run:**

- focus on coupling and dryness control
- verify monomer freshness and solvent quality
- check inert gas dryness and leaks

#### **If deletion impurities are high:**

- focus on capping strength and delivery consistency
- confirm detritylation completion (missed detritylation can also increase deletions)

### If results drift after downtime:

- suspect moisture uptake in lines/solvent
- run purge/conditioning steps
- replace or refresh critical reagents

---

### Best Practices Checklist (Long Oligos)

- Support selected for long-length diffusion/access
- Dry acetonitrile installed and sealed
- Fresh monomers prepared with low moisture exposure
- Activator is fresh and handled correctly
- Gas supply is dry / conditioned
- Capping delivery is strong and consistent
- Conditioning/purge performed after downtime when needed

- [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) <https://www.linkedin.com/company/sierra-biosystems-inc>

Copyright © 2025, Sierra BioSystems