--- title: Annealing Two Single Stranded Oligos To Make Double Stranded DNA (i.e. How to make linkers) description: annealing --- [Skip to content](https://support.sierrabio.com/annealing-two-single-stranded-oligos-to-make-double-stranded-dna-i.e.-how-to-make-linkers#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. [Special Topics](https://support.sierrabio.com/oligonucleotide-chemistry-methods?hsLang=en#special-topics) January 25, 2026 # Annealing Two Single Stranded Oligos To Make Double Stranded DNA (i.e. How to make linkers) ──────────────────────────────────────────────────────── PROCEDURE ──────────────────────────────────────────────────────── 1\) Dissolve oligos in STE buffer    • Buffer composition:      - 10 mM Tris, pH 8.0      - 50 mM NaCl      - 1 mM EDTA    • Note: The presence of salt is required for efficient hybridization.    • Dissolve each oligo at high concentration:      ~10 OD260 units per 100 µL. 2\) Mix complementary strands    • Combine the two strands in EQUAL MOLAR amounts.    • Unequal molar ratios will result in residual single-stranded material. 3\) Heat denaturation and annealing    • Heat mixture to 94 °C.    • Allow to cool gradually to room temperature.    Cooling options:    • Simple sequences:      - Remove from heat and cool on the benchtop at room temperature.    • Sequences with significant hairpin potential:      - Use a gradual cooling step (e.g., water bath or temperature block).      - Heat to 94 °C, then turn off/unplug the device and allow slow cooling. 4\) Storage    • The annealed product will be stable in double-stranded form.    • Store at:      - 4 °C for short-term use      - Frozen for long-term storage ──────────────────────────────────────────────────────── THINGS TO CONSIDER ──────────────────────────────────────────────────────── • Ligation applications:   - A 5′ phosphate may be required.   - This can be added:     - During oligo synthesis (chemical phosphorylation), or     - Post-synthesis using PNK (enzymatic phosphorylation). • Oligo length / cloning applications:   - For longer oligos or cloning workflows, starting with PAGE-purified oligos     is recommended for best results. - [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) - 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