{"id":836,"date":"2012-06-15T21:11:00","date_gmt":"2012-06-15T21:11:00","guid":{"rendered":"https:\/\/stage.idtdna.com\/page\/annealing-oligos"},"modified":"2025-09-15T14:14:56","modified_gmt":"2025-09-15T14:14:56","slug":"annealing-oligonucleotides","status":"publish","type":"post","link":"https:\/\/sgstage.idtdna.com\/page\/support-and-education\/decoded-plus\/annealing-oligonucleotides\/","title":{"rendered":"Annealing oligos protocol"},"content":{"rendered":"

It is sometimes necessary to make double-stranded DNA from single-stranded, complementary oligos. While the oligo annealing (also called DNA annealing, even if this annealing process can also be used for RNA) procedure is fairly straightforward, attention to a few details can help reduce the presence of undesired single-stranded material.<\/p>\n

Oligo annealing protocol<\/strong><\/h2>\n
    \n
  1. \n
      \n
    1. Resuspend\u2014<\/strong>after briefly spinning down each oligonucleotide pellet, dissolve in Duplex Buffer (100 mM potassium acetate; 30 mM HEPES, pH 7.5; available from IDT)\u2014this provides a buffering environment and the salt is necessary for oligonucleotide hybridization. Dissolve each oligo at high concentration (1\u201310 OD260<\/sub>\/100 \u03bcL); see Table 1 for guidelines on resuspension volumes. Heating (up to 94\u00b0C) and vortexing will facilitate resuspension. Confirm the desired concentration by measuring the A260\u00a0<\/sub>of the solution and calculate the concentration<\/a>\u00a0using the extinction coefficient\u00a0(\u03b5) provided on the oligo spec sheet (A =\u00a0\u03b5CL).<\/li>\n
    2. Mix\u2014<\/strong>add the 2 oligo strands together in equal molar amounts. This step is critical to avoid residual single-stranded material.<\/li>\n
    3. Anneal\u2014<\/strong>heat the mixed oligonucleotides to 94\u00b0C for 2 minutes and gradually cool. For many oligos \"cooling\" can be as simple as transferring samples from the heat block or water bath to the bench-top at room temperature. For sequences with significant secondary structure, a more gradual cooling\/annealing step is beneficial. This is easily done by placing the oligo solution in a water bath or heat block and unplugging\/turning off the machine.<\/li>\n
    4. (Optional) Dilute\u2014<\/strong>if needed, dilute the annealed oligonucleotides using Nuclease\u2011Free\u00a0Duplex Buffer<\/a> or 1X\u00a0IDTE\u00a0Buffer<\/a>.<\/li>\n
    5. Store\u2014<\/strong>the resulting product will be in a stable, double-stranded form and we recommend that it is stored at 4\u00b0C or frozen.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n

      Table 1. Recommended resuspension volumes.<\/strong><\/p>\n\n\n\n\n\n\n\n
      Oligo amount (nmol)<\/th>\nVolume for\u00a0100 \u00b5M<\/th>\nVolume for 50\u00a0\u00b5M<\/th>\nVolume for 20\u00a0\u00b5M<\/th>\nVolume for 10\u00a0\u00b5M<\/th>\n<\/tr>\n<\/thead>\n
      10<\/td>\n100 \u00b5L<\/td>\n200 \u00b5L<\/td>\n500 \u00b5L<\/td>\n1 mL<\/td>\n<\/tr>\n
      25<\/td>\n250 \u00b5L<\/td>\n500 \u00b5L<\/td>\n1.25 mL<\/td>\n2.5 mL<\/td>\n<\/tr>\n
      100<\/td>\n1 mL<\/td>\n2 mL<\/td>\n5 mL<\/td>\n10 mL<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

       <\/p>\n

      Tips for annealing your oligos<\/h2>\n

      Avoiding contamination and degradation\u2014<\/strong>if you plan to use the duplex on multiple occasions, divide it into smaller aliquots and store at \u201320\u00b0C. Nuclease\u2011free\u00a0Duplex\u00a0Buffer<\/a> is available from IDT and is certified nuclease-free by testing with IDT RNaseAlert<\/a>\u00ae<\/sup><\/a><\/strong> and DNaseAlert\u2122 Kits<\/a>.<\/p>\n

      Secondary structure\u2014<\/strong>low yields of the expected, annealed product can be caused by secondary structure. Use the OligoAnalyzer\u2122\u00a0program<\/a>\u00a0to determine whether there is significant secondary structure in the oligonucleotides. Problematic annealing can often be resolved by slow cooling, as described in Step 3, above.<\/p>\n

      Ligation\u2014<\/strong>if the double-stranded oligonucleotide product will be used in a ligation reaction, you may need to add 5\u2019-phosphates to the strand ends. These can be added at the time of oligo synthesis (chemical phosphorylation; done by request) or anytime thereafter (before or after annealing) using polynucleotide kinase (enzymatic phosphorylation).<\/p>\n

      Cloning\u2014<\/strong>if the resulting double-stranded DNA fragment will be relatively long (>60 bp), or will be used in cloning, we recommend starting with PAGE-purified oligos (IDT can provide this service).<\/p>\n

      Annealing RNA\u2014<\/strong>the IDT research team also uses this protocol to create siRNA duplexes from single-stranded, complementary RNA oligos.<\/p>\n

      Let us anneal your oligos for you!<\/h2>\n

      For a small fee, IDT will anneal your oligos for you, so that you can proceed with your experiments as soon as your oligos arrive. To request this Duplex Service<\/a>, go to the Duplex Entry page<\/a>.<\/p>\n

      For additional information, contact us<\/a>.<\/p>\n

      RUO23-1663_001.2<\/p>\n","protected":false},"excerpt":{"rendered":"

      It is sometimes necessary to make double-stranded DNA from single-stranded, complementary oligos. While the oligo annealing (also called DNA annealing, even if this annealing process can also be used for RNA) procedure is fairly straightforward, attention to a few details can help reduce the presence of undesired single-stranded material. Oligo annealing protocol Resuspend\u2014after briefly spinning […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"ct_builder_shortcodes":"","ct_template_type":"","ct_parent_template":0,"inline_featured_image":false,"footnotes":""},"class_list":["post-836","post","type-post","status-publish","format-standard","hentry"],"acf":[],"yoast_head":"\nAnnealing Oligos Protocol | IDT<\/title>\n<meta name=\"description\" content=\"Follow this step-by-step oligo annealing protocol to efficiently create double-stranded DNA from single-stranded complementary oligonucleotides.\" \/>\n<meta name=\"robots\" content=\"follow\" \/>\n<link rel=\"canonical\" href=\"https:\/\/sgstage.idtdna.com\/page\/support-and-education\/decoded-plus\/annealing-oligonucleotides\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" 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