{"id":12907,"date":"2019-09-25T23:02:49","date_gmt":"2019-09-25T23:02:49","guid":{"rendered":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/?p=12907"},"modified":"2021-03-18T10:33:34","modified_gmt":"2021-03-18T10:33:34","slug":"pcr-primer-design-tips","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/pcr-primer-design-tips\/","title":{"rendered":"PCR Primer Design Tips"},"content":{"rendered":"

Designing oligonucleotides and making sure that you have the right parameters for your oligo<\/a> is an important step in securing results, especially in PCR Primer Design<\/a>. In order to achieve successful DNA amplification, it\u2019s important to start off with the right primer.<\/p>\n

<\/p>\n

What makes a good primer?<\/h2>\n

Here are some guidelines for designing your PCR primers<\/a>:<\/p>\n

    \n
  1. Aim for the GC content to be between 40 and 60% with the 3\u2019 of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer. Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation.<\/li>\n
  2. A good length\u00a0for\u00a0PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.<\/li>\n
  3. Try\u00a0to make\u00a0the melting temperature (Tm) of the primers between 65\u00b0C and 75\u00b0C, and within 5\u00b0C of each other. Because the Tm is dependent on the length, it\u2019s important to keep primers on the shorter end. The bases also impact the Tm, G and C result in higher melting temperatures than A and T. If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.<\/li>\n
  4. Typically,\u00a03 to 4 nucleotides are added 5 \u2019of the restriction enzyme site in the primer to allow\u00a0for efficient cutting.<\/li>\n
  5. Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT-rich domains.<\/li>\n
  6. Try\u00a0to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or\u00a0ATATATAT).<\/li>\n
  7. Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.<\/li>\n
  8. If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.<\/li>\n
  9. If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.<\/li>\n
  10. If you are using the primers for a PCR reaction to be used in Invitrogen TOPO cloning, the primers should not have a phosphate modification.<\/li>\n<\/ol>\n

     <\/p>\n

    For Research Use Only. Not for Use in Diagnostic Procedures.<\/p>\n","protected":false},"excerpt":{"rendered":"

    Designing oligonucleotides and making sure that you have the right parameters for your oligo is an important step in securing results, especially in PCR Primer Design. In order to achieve successful DNA amplification, it\u2019s important to start off with the right primer. What makes a good primer? Here are some guidelines for designing your PCR<\/p>\n","protected":false},"author":120,"featured_media":12909,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[108],"tags":[422],"division":[],"class_list":{"0":"post-12907","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-lab-tips-and-tricks","8":"tag-pcr-workflow","9":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"\nPCR Primer Design Tips - Behind the Bench<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.thermofisher.com\/blog\/behindthebench\/pcr-primer-design-tips\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"PCR Primer Design Tips\" \/>\n<meta property=\"og:description\" content=\"Designing oligonucleotides and making sure that you have the right parameters for your oligo is an important step in securing results, especially in PCR Primer Design. 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However, the effectiveness of real-time PCR can be increased by an approach called multiplex qPCR.\u2026","rel":"","context":"In "General"","block_context":{"text":"General","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/general\/"},"img":{"alt_text":"","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/12\/Web-header-900-x-600-custom-web-page-size-feature-pods.png?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/12\/Web-header-900-x-600-custom-web-page-size-feature-pods.png?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/12\/Web-header-900-x-600-custom-web-page-size-feature-pods.png?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/12\/Web-header-900-x-600-custom-web-page-size-feature-pods.png?resize=700%2C400&ssl=1 2x"},"classes":[]},{"id":19447,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/philadelphia-chromosome-like-all-digital-pcr\/","url_meta":{"origin":12907,"position":2},"title":"Unlocking Cancer\u2019s Genetic Secrets with Digital PCR","author":"Behind The Bench Staff","date":"February 27, 2026","format":false,"excerpt":"In this episode of Absolute Gene-ius, we explore how digital PCR (dPCR) is transforming leukemia research with Valeria Rangel, a PhD candidate at the University of California, Irvine. 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The benefits are clear: reduced sample consumption, lower costs, faster workflows, and more data from every experiment. However, designing successful multiplex assays isn't always straightforward. Traditionally, identifying problematic assay combinations required extensive wet-lab\u2026","rel":"","context":"In "Lab Tips and Tricks"","block_context":{"text":"Lab Tips and Tricks","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/lab-tips-and-tricks\/"},"img":{"alt_text":"For Research Use Only. Not for use in diagnostic procedures. \u00a9 2026 Thermo Fisher Scientific Inc. All rights reserved. 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