{"id":117,"date":"2022-07-21T13:43:53","date_gmt":"2022-07-21T17:43:53","guid":{"rendered":"https:\/\/research.ncsu.edu\/gsl\/?page_id=117"},"modified":"2025-12-10T20:09:26","modified_gmt":"2025-12-11T01:09:26","slug":"genotyping-guidelines","status":"publish","type":"page","link":"https:\/\/research.ncsu.edu\/gsl\/dna-sequencing\/genotyping-guidelines\/","title":{"rendered":"Fragment Genotyping Guidelines"},"content":{"rendered":"\n\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-0\"><strong>Genotyping Services<\/strong><\/h2>\n\n\n\n<p>The GSL only accepts ready-to-load 96-well plates, submitted through our&nbsp;<a href=\"https:\/\/research.ncsu.edu\/gsl\/sanger-sequencing\/how-to-submit-a-project\/\">online system<\/a>.<\/p>\n\n\n\n<p>All plates submitted must be 96-well semi skirted PCR Plates (available for purchase at the GSL). One of the following plate types is&nbsp;<em>required<\/em>&nbsp;for plate submission:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>USA Scientific: Part Number 1402-9200<\/li>\n\n\n\n<li>Genesee Scientific: Part Number 27-408<\/li>\n<\/ul>\n\n\n\n<p>Users are responsible for preparation and clean-up of their samples prior to submission of the plate. We perform the loading of the capillary sequencers.<\/p>\n\n\n\n<p>All Genotyping plates are processed on an Applied Biosystems 3730xl DNA Analyzer. Genotyping on the 3730xl has a wide range of applications including AFLP, Microsatellite, SNP analysis, etc. The 3730xl is optimized for a 5-dye set (Dye set 33 Filter set G5). The dyes in this set include 6-FAM, VIC, NED, PET, and LIZ size standard. The 5-dye set has increased the multiplexing capabilities.<\/p>\n\n\n<div class=\"has-custombg-one-background-color wp-block-ncst-button-spotlight\">\n    <div class=\"button-spotlight__container\">\n          <div class=\"button-spotlight__icon-container ncst-icon\">\n        <svg viewBox=\"0 0 24 24\" fill=\"none\" xmlns=\"http:\/\/www.w3.org\/2000\/svg\">\n<path d=\"M23.9999 1.9664H22.2463V22.0336H23.9999V1.9664Z\" fill=\"currentColor\"\/>\n<path d=\"M1.30219 9.89015H0V14.1094H1.30219V9.89015Z\" fill=\"currentColor\"\/>\n<path d=\"M21.4787 4.19952L2.07056 10.5141V13.4859L6.38306 14.8889C6.35454 14.9524 6.32966 15.0175 6.30853 15.0839C6.15766 15.5612 6.20229 16.0789 6.43261 16.5234C6.66293 16.9679 7.06014 17.3028 7.53712 17.4548L9.77962 18.1664C10.257 18.3166 10.7743 18.2716 11.2186 18.0412C11.6628 17.8109 11.9978 17.414 12.1501 16.9373C12.167 16.8848 12.1801 16.8319 12.1918 16.7789L21.4782 19.8005L21.4787 4.19952ZM11.2656 16.6566C11.1876 16.8984 11.0173 17.0997 10.7917 17.2167C10.5661 17.3336 10.3034 17.3567 10.0609 17.2809L7.81837 16.5698C7.57594 16.4925 7.37407 16.3223 7.25702 16.0963C7.13997 15.8704 7.11731 15.6073 7.19399 15.3647C7.21538 15.3004 7.24334 15.2385 7.27743 15.18L11.3007 16.4887C11.2942 16.5457 11.2825 16.6018 11.2656 16.6566Z\" fill=\"currentColor\"\/>\n<\/svg>\n\n      <\/div>\n        <div class='button-spotlight__content-container'>\n      <h2 class=\"button-spotlight__heading\">Sunsetting Select Services<\/h2>\n              <p class=\"button-spotlight__support\">Effective Feb. 2, 2026, the GSL will no longer offer Sanger sequencing or fragment genotyping, as part of a broader effort to streamline our services.<\/p>\n            \n<div class=\"is-style-secondary wp-block-ncst-button\">\n      <a\n        class=\"ncst-block__button-link btn\"\n        href=\"https:\/\/research.ncsu.edu\/gsl\/dna-sequencing\/sanger-sequencing-fragment-genotyping\/sunsetting-faq\/\"\n        data-ncst-lightbox=\"false\"\n                      >\n                  <span class=\"text\">Learn more<\/span><span class=\"arrow-indicator\"><svg class=\"wolficon wolficon-arrow-right-bold\" role=\"img\"  aria-hidden=\"true\">\n\t\t\t\n\t\t\t<use xlink:href=\"#wolficon-arrow-right-bold\">\n\t\t<\/svg><\/span>\n              <\/a>\n    <\/div>\n  \n\n    <\/div>\n  <\/div>\n<\/div>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-1\">Genotyping Steps<\/h2>\n\n\n\n<ol class=\"wp-block-list\">\n<li>DNA extraction via commercial kits, phenol extraction etc.<\/li>\n\n\n\n<li>Quantify DNA using agarose electrophoresis, spectrophotometry, etc.<\/li>\n\n\n\n<li>Perform PCR with dye labeled primers<\/li>\n\n\n\n<li>Multiplex by combining PCR products<\/li>\n\n\n\n<li>Set-up plate with PCR products, Hi-Di Formamide, and size standard<\/li>\n\n\n\n<li>Submit plate and plate record<\/li>\n\n\n\n<li>Data retrieval<\/li>\n<\/ol>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-2\">Genotyping Reaction Setup<\/h2>\n\n\n\n<ul class=\"wp-block-list\">\n<li>PCR Template: Optimized by user<\/li>\n\n\n\n<li>Size Standard: 0.5 uL per reaction<\/li>\n\n\n\n<li>High-Di Formamide: Amount needed to bring final reaction volume to 10 uL.<\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-3\">PCR Template<\/h2>\n\n\n\n<p>Users are required to optimize the amount of template best for their samples.&nbsp; If the user desires to multiplex, it is best to run the PCR reactions separately and then combine them in the final plate.&nbsp; You can have up to 4 different PCR products in one well; one for each of the available dyes.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-4\">Size Standard<\/h2>\n\n\n\n<p>LIZ-600 is the available size standard for the dye set optimized on the 3730xl sequencer.&nbsp; Use 0.5 uL per reaction.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-5\">Hi-Di Formamide<\/h2>\n\n\n\n<p>Users will need to purchase highly de-ionized formamide from Life Technologies.&nbsp; It is important to get this grade of formamide because others will affect the sequencing run.&nbsp; Use the formamide to bring the final volume of the reaction to 10uL.&nbsp; Empty wells should be filled with 10uL of water.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-6\">Plate Record Submission<\/h2>\n\n\n\n<p>You must attach a plate record to your&nbsp;<a href=\"https:\/\/research.ncsu.edu\/gsl\/sanger-sequencing\/how-to-submit-a-project\/\">online submission<\/a>&nbsp;(templates are available in the submission portal). The plate record must match the name that is physically on your plate. The plate name and sample names should follow the rules below:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Must be 10 characters or less<\/li>\n\n\n\n<li>No spaces<\/li>\n\n\n\n<li>No unusual characters; you may use underscores __<\/li>\n\n\n\n<li>Name all wells, if needed use blank, empty, water, etc.<\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-7\">To make a plate record<\/h2>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Download the plate submission sheet (through the&nbsp;<a href=\"https:\/\/research.ncsu.edu\/gsl\/sanger-sequencing\/how-to-submit-a-project\/\">submission portal<\/a>) and list your sample names in 1 single column. The order should go DOWN your plate columns, NOT across.<\/li>\n\n\n\n<li>Save the plate record as a tab delineated text file (.txt)<\/li>\n\n\n\n<li>Upload the file to your request in the submission system.<\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\" id=\"autoheading-8\">Data Retrieval<\/h2>\n\n\n\n<p>Data will be emailed from the&nbsp;<a href=\"mailto:GSLNCSU@gmail.com\">GSLNCSU@gmail.com<\/a>&nbsp;account.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Genotyping Services The GSL only accepts ready-to-load 96-well plates, submitted through our&nbsp;online system. All plates submitted must be 96-well semi skirted PCR Plates (available for purchase at the GSL). One&hellip;<\/p>\n","protected":false},"author":1127,"featured_media":0,"parent":42,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"ncst_custom_author":"","ncst_show_custom_author":false,"ncst_dynamicHeaderBlockName":"ncst\/floating-box-header","ncst_dynamicHeaderData":"{\"backgroundColor\":\"red_400\",\"boxPosition\":\"left\",\"ctaNum\":\"one\",\"useCTA\":false,\"imageID\":54,\"imageURL\":\"https:\/\/research.ncsu.edu\/gsl\/files\/2022\/07\/about-dna.jpg\",\"imageAlt\":\"blue dna\"}","ncst_content_audit_freq":"","ncst_content_audit_date":"","ncst_content_audit_display":false,"ncst_backToTopFlag":"","footnotes":""},"tags":[],"class_list":["post-117","page","type-page","status-publish","hentry"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.5 - https:\/\/yoast.com\/wordpress\/plugins\/seo\/ -->\n<title>Fragment Genotyping Guidelines - Genomic Sciences Laboratory<\/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:\/\/research.ncsu.edu\/gsl\/dna-sequencing\/genotyping-guidelines\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Fragment Genotyping Guidelines - Genomic Sciences Laboratory\" \/>\n<meta property=\"og:description\" content=\"Genotyping Services The GSL only accepts ready-to-load 96-well plates, submitted through our&nbsp;online system. 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