--- title: Optimizing Protocols in K&A or Shasta Software description: This article goes over how to optimize protocols for K&A and Shasta --- [Skip to content](https://support.sierrabio.com/optimizing-protocols-in-ka-or-shasta-software#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. [Troubleshooting & Errors](https://support.sierrabio.com/troubleshooting-errors?hsLang=en) 3. [Chemistry and Protocol-Related Issues](https://support.sierrabio.com/troubleshooting-errors?hsLang=en#chemistry-and-protocol-related-issues) August 1, 2025 # Optimizing Protocols in K&A or Shasta Software ## Optimizing your protocol can improve coupling efficiency, increase yields, reduce failed columns, and make your workflow more consistent The **single most important tip** is: > **Always watch 1 or 2 base cycle and adjust based on what you see.** > > No amount of protocol tweaking will help if you’re not visually confirming how your reagents behave during the run. Watching a cycle will tell you: > > - Whether deblock is fully removing trityl > - If washes are clearing the previous reagent > - If coupling looks strong and stable > - If priming is working effectively (especially for Shasta) Dedicate columns if needed to observe and optimize — it will save you time, money, and frustration later. --- #### **1. Coupling Times** Coupling time is a major factor in synthesis success: - **DNA:** Minimum **60 seconds** - **RNA:** Minimum **6 minutes** - **Dyes / Complicated Monomers:** **6 to 15 minutes** **General rule:** The greater the molecular size or steric hindrance, the more coupling time and possibly coupling repetitions you’ll need. For longer oligos: - Add **1 extra coupling** for every **40–60 bases**. This can be done through the meta protocol ![](https://support.sierrabio.com/hs-fs/hubfs/image-png-Aug-01-2025-02-33-45-3775-PM.png?width=399&height=304&name=image-png-Aug-01-2025-02-33-45-3775-PM.png) For **large-scale syntheses (≥25 μmol)**: - Increase amidite concentration to maintain high coupling efficiency — especially for bulky or low-reactivity monomers. --- #### **2. Deblock** Deblock steps remove the trityl group from the 5’ end: - **DCA:** 15–60 seconds - **TCA:** 10–40 seconds Tips: - **Watch the cycle** to ensure all trityl is removed. - Adjust deblock repetitions based on what you see. - For long oligos, add **1 extra deblock** for every **40–60 bases**. --- #### **3. Wash Steps** - Washes can be short **if** they are enough to remove all traces of the previous reagent. - Watching the cycle will confirm this — don’t rely on time alone. **DEA Wash:** - Use **only in the final steps of synthesis**. - Main purpose: **Prevents N3 cyanoethylation**. - Especially useful for RNA or sensitive modifications. --- #### **4. Sulfurization & Oxidation Order** Correct step order preserves backbone integrity: - **Sulfurization:** Always **before capping** — prevents acetic anhydride from interfering. - **Oxidation:** Best **after capping**. Add an **extra capping step after oxidation** to help remove moisture from oxidizer. **If using mixed sulfur and oxygen backbones:** - **PS → Cap → Ox → Cap** **![](https://api.hubspot.com/filemanager/api/v2/files/193680273842/signed-url-redirect?portalId=47834229)** - Oxidizer-only: **Cap → Ox → Cap** - Sulfur-only: **Sulfur → Cap → Cap** --- #### **5. Supports** Steric hindrance increases as the oligo grows: - **Long oligos (60+ bases) / bulky groups  :** Use **2000 Å CPG** for higher efficiency. - **Polystyrene supports** swell and are **not recommended for K&A**. - **Standard support** increases yield and does not require heat for cleavage. 💡 **For standard supports:** A small percentage of Standard Support can become unfunctionalized. Add a **capping step during initialization** to block unlinked sites and prevent N-1 products — especially important with bulky 3’ modifiers. --- #### **6. Moisture Control** Even trace moisture reduces coupling efficiency. - Use fresh, anhydrous ACN and amidites. - Add trap packs (molecular sieves) to ACN, activator, and amidites at least **1 hour before synthesis**. - Use in-line drying filters for argon/nitrogen. - Monitor room humidity near the instrument. --- #### **7. K&A vs Shasta Instrument Considerations** ### **K&A – Trityl Monitor** - Use trityl monitor to track coupling efficiency. - High initial V2 values can make readings unreliable — often a timing issue, not a chemistry failure. - Always confirm trityl data by watching the cycle. - Check out our [Understanding Trityl Monitor](https://47834229.hs-sites.com/ka-understanding-trityl-monitors?hsLang=en) article for more information --- ### **Shasta – Crystallization & Priming** Shasta’s open column design makes crystallization common. - **Prime infrequently used tips every  every cycle** — at least **1 prime**, preferably **2**.![](https://support.sierrabio.com/hs-fs/hubfs/image-png-Aug-01-2025-02-31-32-5943-PM.png?width=488&height=415&name=image-png-Aug-01-2025-02-31-32-5943-PM.png) - Ensure chamber is **fully sealed** to reduce solvent evaporation and crystallization. - **O‑methyl‑U and LNA‑C**: prone to crashing out. Use **10–20% DMF or THF** as cosolvent. - Keep **dummy “poly” sequences** for routine priming (e.g., Poly O‑methyl‑U) — ensures amidites are primed every cycle, reducing random column failures. --- #### **8. Saving Protocol Versions** - Each change should be saved as a new protocol (e.g., *RNA\_Long\_v2*). - Document what was changed and why. - Compare results to your baseline runs. --- #### **When to Contact Support** If, after optimization, you still see: - Severe yield loss - Inconsistent coupling - Random unexplained failures Contact our service team with your run files and protocol settings. - [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