{"id":8904,"date":"2015-08-12T18:37:29","date_gmt":"2015-08-12T18:37:29","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/behindthebench\/?p=8904"},"modified":"2018-10-18T23:38:46","modified_gmt":"2018-10-18T23:38:46","slug":"designing-pcr-and-sanger-sequencing-primers-seq-it-out-5","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/designing-pcr-and-sanger-sequencing-primers-seq-it-out-5\/","title":{"rendered":"Designing PCR and Sanger Sequencing Primers\u2013 Seq It Out #5"},"content":{"rendered":"<p>Primer design is like art. There is more than one design to cover the region of interest. Are you an \u201cArtist\u201d?<\/p>\n<p>Primers are crucial to the success of target amplification and subsequent sequencing in PCR and Sanger sequencing workflows.<\/p>\n<p><iframe loading=\"lazy\" width=\"760\" height=\"428\" src=\"https:\/\/www.youtube.com\/embed\/P6Nm9Vxyzj0?feature=oembed\" frameborder=\"0\" allow=\"accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture\" allowfullscreen><\/iframe><\/p>\n<p>Let\u2019s take a look at our lab book:<\/p>\n<p>In the typical <a class=\"jive-link-external-small\" href=\"http:\/\/www.lifetechnologies.com\/us\/en\/home\/life-science\/sequencing\/sanger-sequencing\/sanger-dna-sequencing.html?socid=social_btb\" rel=\"nofollow\">Sanger sequencing workflow <\/a>from genomic DNA, one needs to first amplify the target by PCR, and then subsequently run the Sanger sequencing reaction.\u00a0 If you start from purified plasmid DNA, one only needs to run the Sanger sequencing reaction. PCR amplification requires 2 primers from opposite strands that determine the region of sequence amplified in the forward and reverse direction.<\/p>\n<p>Sanger sequencing differs from PCR in that only a single primer is used in the reaction. Typically, for a given PCR fragment, two Sanger sequencing reactions are set up, one for sequencing the forward strand, the other one for sequencing the reverse strand. Primer design is an important aspect relating to many forms of PCR including basic PCR, fragment analysis, quantitative analysis and Sanger sequencing..<\/p>\n<p>Here are a few things to keep in mind when designing your own primers.<\/p>\n<ol>\n<li>Primer length should be in the range of 18 to 22 bases.<\/li>\n<li>The primer should have GC content of 50% to 55%.<\/li>\n<li>Primers should have a GC-lock on the 3\u2019 end.<\/li>\n<li>The melting temperature of any good primer should be in the range of 50OC to 55OC.<\/li>\n<li>The primer should not include poly base regions.<\/li>\n<li>Four or more bases that compliment either direction of the primer should be avoided.<\/li>\n<\/ol>\n<p>In addition, there are some PCR specific guidelines to help you design good PCR primers. These guidelines can be found on our website.<\/p>\n<p>Since primer design is not easy.\u00a0 There is risk of designing the wrong primers which could be costly in your experiments. You may be asking: Is there an easier way?\u00a0 The answer is Yes, and Thermo Fisher Scientific has a free tool to help you out. <a class=\"jive-link-external-small\" href=\"http:\/\/www.lifetechnologies.com\/us\/en\/home\/life-science\/sequencing\/sanger-sequencing\/pre-designed-primers-pcr-sanger-sequencing.html?socid=social_ABSeq\" rel=\"nofollow\">Primer Designer tool<\/a> is a free PCR\/Sanger primer online search tool that includes over 600,000 primer pairs covering the human exome and human mitochondrial genome.\u00a0 You can choose the range of amplicon length for your sample and your research interest to optimize it for your experiment.<\/p>\n<p>If you are doing NGS confirmation with your capillary electrophoresis genetic analyzer, this tool can really simplify your workflow. You can use this online by uploading your .vcf file, or if you have an Ion Torrent sequencer, even better, as there is a seamless integration with your ion reporter software to upload your data into this Primer Designer tool.\u00a0 Now you can find the right primers with just a few simple clicks.<\/p>\n<p>I hope this video was helpful on primer design, and I am sure you\u2019ll have more questions.<\/p>\n<p>Submit your question at <a class=\"jive-link-external-small\" href=\"http:\/\/www.lifetechnologies.com\/us\/en\/home\/products-and-services\/promotions\/ask.html?socid=social_ABSeq\" rel=\"nofollow\">http:\/\/www.thermofisher.com\/ask<\/a> and subscribe to our channel to see more videos like this.<\/p>\n<p>And remember, when in doubt, just Seq It Out<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Primer design is like art. There is more than one design to cover the region of interest. Are you an \u201cArtist\u201d? Primers are crucial to the success of target amplification and subsequent sequencing in PCR and Sanger sequencing workflows. Let\u2019s take a look at our lab book: In the typical Sanger sequencing workflow from genomic<\/p>\n","protected":false},"author":117,"featured_media":8905,"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":[17,36,176,74],"division":[],"class_list":{"0":"post-8904","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-lab-tips-and-tricks","8":"tag-applied-biosystems","9":"tag-capillary-electrophoresis","10":"tag-seqitout","11":"tag-sequencing","12":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.8 (Yoast SEO v27.8) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>Designing PCR and Sanger Sequencing Primers\u2013 Seq It Out #5 - 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\/designing-pcr-and-sanger-sequencing-primers-seq-it-out-5\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Designing PCR and Sanger Sequencing Primers\u2013 Seq It Out #5\" \/>\n<meta property=\"og:description\" content=\"Primer design is like art. There is more than one design to cover the region of interest. Are you an \u201cArtist\u201d? Primers are crucial to the success of target amplification and subsequent sequencing in PCR and Sanger sequencing workflows. 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Today\u2019s labs are not only looking for accuracy\u2014they need speed, flexibility, and reliable\u2026","rel":"","context":"In &quot;General&quot;","block_context":{"text":"General","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/general\/"},"img":{"alt_text":"DNA sequencing sample tube with DNA helix and sequencing chromatogram representing Sanger sequencing workflow.","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/ChatGPT-Image-Aug-5-2026-09_44_12-AM.png?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/ChatGPT-Image-Aug-5-2026-09_44_12-AM.png?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/ChatGPT-Image-Aug-5-2026-09_44_12-AM.png?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/ChatGPT-Image-Aug-5-2026-09_44_12-AM.png?resize=700%2C400&ssl=1 2x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/ChatGPT-Image-Aug-5-2026-09_44_12-AM.png?resize=1050%2C600&ssl=1 3x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/ChatGPT-Image-Aug-5-2026-09_44_12-AM.png?resize=1400%2C800&ssl=1 4x"},"classes":[]},{"id":19888,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/streptococcus-pneumoniae-serotype-identification-qpcr-nested-pcr\/","url_meta":{"origin":8904,"position":1},"title":"qPCR, Nested PCR, and the Search for Bacterial Serotypes","author":"Ashleigh Barlow","date":"August 5, 2026","format":false,"excerpt":"Article Summary In this episode of Absolute Gene-ius, Donna Paznar, former Head of Molecular Infection Diagnostics at the Medical University of Vienna, discusses her research on Streptococcus pneumoniae and its impact across the Austrian population. She explains why children can act as asymptomatic reservoirs, why tracking bacterial serotypes matters, and\u2026","rel":"","context":"In &quot;Infectious Diseases&quot;","block_context":{"text":"Infectious Diseases","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/infectious-diseases\/"},"img":{"alt_text":"Donna Paznar discussing molecular methods for identifying Streptococcus pneumoniae serotypes.","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/Paznar_Headshot_Jordan-Ruggieri-scaled.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/Paznar_Headshot_Jordan-Ruggieri-scaled.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/Paznar_Headshot_Jordan-Ruggieri-scaled.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/Paznar_Headshot_Jordan-Ruggieri-scaled.jpg?resize=700%2C400&ssl=1 2x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/Paznar_Headshot_Jordan-Ruggieri-scaled.jpg?resize=1050%2C600&ssl=1 3x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/Paznar_Headshot_Jordan-Ruggieri-scaled.jpg?resize=1400%2C800&ssl=1 4x"},"classes":[]},{"id":20004,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/a-guide-to-nucleic-acid-quality-control-dna-and-rna-purity\/","url_meta":{"origin":8904,"position":2},"title":"A Guide to Nucleic Acid Quality Control: DNA and RNA Purity","author":"Marlene Gasdia-Cochrane","date":"September 22, 2026","format":false,"excerpt":"Article Summary Nucleic acid quality control helps researchers evaluate DNA and RNA concentration and purity before downstream applications such as PCR, qPCR, and next-generation sequencing (NGS). A standardized workflow combining reliable extraction, concentration and purity assessment, application-appropriate quantification, and consistent laboratory practices can help identify sample issues early, improve reproducibility,\u2026","rel":"","context":"In &quot;General&quot;","block_context":{"text":"General","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/general\/"},"img":{"alt_text":"Close-up shot of a dna sequence on a screen, a digital representation of genetic code, used for analysis.","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=700%2C400&ssl=1 2x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=1050%2C600&ssl=1 3x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=1400%2C800&ssl=1 4x"},"classes":[]},{"id":19447,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/philadelphia-chromosome-like-all-digital-pcr\/","url_meta":{"origin":8904,"position":3},"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. Her work focuses on Philadelphia chromosome-like (Ph-like) B-cell acute lymphoblastic leukemia (ALL), a rare, aggressive subtype that disproportionately affects Hispanic populations. By\u2026","rel":"","context":"In &quot;Cancer Research&quot;","block_context":{"text":"Cancer Research","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/cancer-research\/"},"img":{"alt_text":"","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/11\/RANGEL-headshot-1.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/11\/RANGEL-headshot-1.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/11\/RANGEL-headshot-1.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/11\/RANGEL-headshot-1.jpg?resize=700%2C400&ssl=1 2x"},"classes":[]},{"id":19883,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/building-a-better-sample-qc-workflow-from-extraction-to-sequencing\/","url_meta":{"origin":8904,"position":4},"title":"Building a Better Sample QC Workflow: From Extraction to Sequencing","author":"Marlene Gasdia-Cochrane","date":"August 25, 2026","format":false,"excerpt":"Article Summary Successful genomics experiments don't begin with sequencing\u2014they begin with sample quality. Every downstream application, from PCR to next-generation sequencing (NGS), depends on starting with nucleic acids that are accurately quantified and free from contaminants that could compromise results. Establishing a consistent quality control workflow helps laboratories improve reproducibility\u2026","rel":"","context":"In &quot;General&quot;","block_context":{"text":"General","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/general\/"},"img":{"alt_text":"A pipette drops a liquid into a petri dish with a dna sequence background, science concept. It suggests medical or genetic research.","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=700%2C400&ssl=1 2x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=1050%2C600&ssl=1 3x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=1400%2C800&ssl=1 4x"},"classes":[]},{"id":19345,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/precision-oncology-diagnostics\/","url_meta":{"origin":8904,"position":5},"title":"The Expanding Role of Molecular Diagnostics in Precision Oncology","author":"Behind The Bench Staff","date":"January 13, 2026","format":false,"excerpt":"https:\/\/www.youtube.com\/watch?v=F6FfHIizJBY For Dr. Harald Bartsch, a clinical pathologist in southern Bavaria, molecular diagnostic tools have become an indispensable part of his arsenal. \u201cI believe molecular diagnostics play a crucial role in modern oncology, and I see this as the direction everything is headed,\u201d says Dr. Bartsch. 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