[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"detail-sidebar-cat-1-en-105":3,"doc-seo-242281-105":53,"doc-detail-242281-en":127},{"code":4,"msg":5,"data":6},0,"success",[7,14,19,24,29,34,39,44,49],{"id":8,"doc_module":9,"doc_module_name":10,"category_name":11,"show_sort_weight":12,"slug":13},11,1,"Template","Presentations",90,"presentations",{"id":15,"doc_module":9,"doc_module_name":10,"category_name":16,"show_sort_weight":17,"slug":18},12,"Resumes",80,"resumes",{"id":20,"doc_module":9,"doc_module_name":10,"category_name":21,"show_sort_weight":22,"slug":23},14,"Invoices",70,"invoices",{"id":25,"doc_module":9,"doc_module_name":10,"category_name":26,"show_sort_weight":27,"slug":28},15,"Posters",60,"posters",{"id":30,"doc_module":9,"doc_module_name":10,"category_name":31,"show_sort_weight":32,"slug":33},16,"Social Media",50,"social-media",{"id":35,"doc_module":9,"doc_module_name":10,"category_name":36,"show_sort_weight":37,"slug":38},17,"Forms",40,"forms",{"id":40,"doc_module":9,"doc_module_name":10,"category_name":41,"show_sort_weight":42,"slug":43},18,"Letters",30,"letters",{"id":45,"doc_module":9,"doc_module_name":10,"category_name":46,"show_sort_weight":47,"slug":48},21,"Paper Templates",5,"papers-templates",{"id":50,"doc_module":9,"doc_module_name":10,"category_name":51,"show_sort_weight":4,"slug":52},158,"General","general-158",{"code":4,"msg":54,"data":55},"ok",{"site_id":56,"language":57,"slug":58,"title":59,"keywords":60,"description":61,"schema_data":62,"social_meta":120,"head_meta":122,"extra_data":124,"updated_unix":126},105,"en","bar-seq-library-preparation-and-pooling-primer-preparation","Bar-Seq Library Preparation and Pooling - Primer Preparation","","Bar-Seq Library Preparation and Pooling - Primer Preparation provides a controlled primer-handling workflow to prevent cross-contamination between primers before loading onto automation. The protocol minimizes contamination risk for primers used in the 1st PCR, which attach multiplexing tags to BarSeq DNA amplicons, and the 2nd PCR, which attach sequencing flow cell adapters. Following a “one at a time” principle, it specifies how to open primer stocks once, prepare aliquots sequentially, store primer mixes at defined temperatures, and includes guidance on cleaning and pacing to complete the full set efficiently.",{"@graph":63,"@context":119},[64,80,102],{"@type":65,"itemListElement":66},"BreadcrumbList",[67,71,74,77],{"item":68,"name":69,"@type":70,"position":9},"https://docshare.wps.com","Home","ListItem",{"item":72,"name":10,"@type":70,"position":73},"https://docshare.wps.com/template/",2,{"item":75,"name":46,"@type":70,"position":76},"https://docshare.wps.com/template/paper-templates/",3,{"item":78,"name":59,"@type":70,"position":79},"https://docshare.wps.com/template/bar-seq-library-preparation-and-pooling-primer-preparation/242281/",4,{"url":78,"name":59,"@type":81,"image":82,"author":87,"headline":59,"publisher":90,"fileFormat":93,"inLanguage":57,"description":61,"dateModified":94,"datePublished":95,"encodingFormat":93,"isAccessibleForFree":96,"interactionStatistic":97},"DigitalDocument",{"url":83,"@type":84,"width":85,"height":86},"https://docshare.wps.com/thumbnails/bar-seq-library-preparation-and-pooling-primer-preparation/242281.png","ImageObject",442,249,{"name":88,"@type":89},"Stanford","Person",{"url":68,"name":91,"@type":92},"DocShare","Organization","application/pdf","2026-10-16","2026-09-12",true,{"@type":98,"interactionType":99,"userInteractionCount":101},"InteractionCounter",{"@type":100},"ViewAction",8,{"@type":103,"mainEntity":104},"FAQPage",[105,111,115],{"name":106,"@type":107,"acceptedAnswer":108},"What is the primary purpose of this primer preparation protocol?","Question",{"text":109,"@type":110},"To ensure no cross-contamination between primers before they are loaded onto the automation system. It targets contamination risks across both the 1st PCR and 2nd PCR primer sets.","Answer",{"name":112,"@type":107,"acceptedAnswer":113},"What does the protocol mean by the “one at a time” principle?",{"text":114,"@type":110},"Only one primer stock should be opened at a time, and only one primer’s aliquots should be prepared or manipulated at a time. Primer tubes are opened only once after receiving them from the vendor.",{"name":116,"@type":107,"acceptedAnswer":117},"How are primers for the 1st PCR and 2nd PCR organized?",{"text":118,"@type":110},"The 1st PCR uses multiplexing tag primers covering rows (forward) and columns (reverse) of a 96-well plate to map samples precisely. The 2nd PCR uses universal tags (one forward and one reverse) to enable attachment to the Illumina flow cell.","https://schema.org",{"og:url":78,"og:type":121,"og:title":59,"og:site_name":91,"og:description":61},"article",{"robots":123,"canonical":78},"index,follow",{"doc_id":125,"site_id":56},242281,1790364894,{"code":4,"msg":5,"data":128},{"doc_id":125,"user_id":129,"nickname":88,"user_avatar":130,"doc_module":9,"category_id":45,"category_name":46,"doc_title":59,"doc_description":61,"doc_content":131,"file_id":132,"file_url":133,"file_type":134,"file_size":135,"view_count":101,"is_deleted":4,"is_public":9,"is_downloadable":9,"audit_status":9,"page_count":25,"language":136,"language_code":57,"site_id":56,"html_lang":57,"table_of_contents":137,"faqs":138,"seo_title":139,"seo_description":61,"update_tm":140,"read_time":47},2336477552062,"https://ap-avatar.wpscdn.com/davatar_994ba38a5ba835b3df7d355c54d3ed8d","May 17, 2025  \n Bar-Seq Library Preparation and Pooling: Primer Preparation  \nDOI  \n[https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1](https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1)[ ](https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1)Nina Alperovich1, David Ross1  \n1 NIST  \nOpen Datasets Initiative  \nAlign to Innovate  \nCreate & collaborate more with a free account  \nEdit and publish protocols, collaborate in communities, share insights through comments, and track progress with run records.  \nCreate free account  \nDOI: [https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1](https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1)  \nProtocol Citation: Nina Alperovich, David Ross 2025. Bar-Seq Library Preparation and Pooling: Primer Preparation. protocols. io  \n[https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1](https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1)  \nLicense: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited  \nProtocol status: Working  \nWe use this protocol and it 's working Created: December 12, 2024  \n[protocols.io](protocols.io |)[ |](protocols.io |) [https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1](https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1) May 16, 2025 1/15  \nLast Modified: May 17, 2025  \nProtocol Integer ID: 115039  \nKeywords: primer preparation this protocol, 1st pcr in the barseq protocol, primer preparation, primers in the 2 nd pcr, primers for the 1st pcr, 1st pcr primer, barseq protocol, tags to the barseq dna amplicon, barseq dna amplicon, similar naming strategy to the 1st pcr primer, seq automation instruction, aliquots for each primer, seq library preparation, sequencing flow cell adapter, reverse primer, pooling protocol, tube of primer, full set of primer, aliquots of primer, aliquot for the next primer, primer, next primer, forward primer, primer id, corresponding primer id, 1st pcr in row, seq, label as barseq-1 , barseq-1, primer stock, barseq-1-f2 , 1st pcr, 2 nd pcr, pcr, preparation of aliquot, contamination, barseq-2-f, goal of this protocol, multiplexing tag, amplified pcr product, protocol, sequencing, automation instruction  \nDisclaimer  \nThe protocol outlined in this document was created as a part of Growth-based Quantitative SequencingGROQ􀃂Seq) Platform. The GROQ􀃂Seq platform was created under Align to Innovateʼs Open Dataset Initiative. Align to Innovate is a non-profit research organization operating under open science principles with the goal of improving science research with programmable experiments. The Open Datasets Initiative is working to accelerate community-driven science with the use of automated labs to pioneer robust data collection methods and curated, high-fidelity, public biological datasets amenable to machine learning. This work was supported by Align to Innovateʼs Open Datasets Initiative which receives philanthropic funding in part from Griffin Catalyst.  \n[protocols.io](protocols.io |)[ |](protocols.io |) [https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1](https://dx.doi.org/10.17504/protocols.io.n2bvj9n1nlk5/v1) May 16, 2025 2/15  \nAbstract  \nThis protocol is a rigorous way to ensure no cross-contamination between primers used in the Bar-Seq Library Preparation and Pooling protocols (see Bar-Seq Automation Instructions & User Instructions) . The goal of this protocol is to minimize the opportunities for cross-contamination between the primers for the 1st PCR (which attach the sample multiplexing tags to the BarSeq DNA amplicons) as well as the 2 nd PCR (which attach sequencing flow cell adapters) . To this end, this protocol is designed to make it physically impossible for crosscontamination to occur before the primers are loaded onto the automation system.  \nThe main principle is “one at a timeˮ: only one ","cbCaieUlQWwAMtYx","https://ap.wps.com/l/cbCaieUlQWwAMtYx","pdf",731400,"English","# Abstract\n## Cross-contamination prevention strategy\n## “One at a time” handling principle\n## Primer aliquoting and storage\n## Primer set design (1st PCR and 2nd PCR)","[{\"question\":\"What is the primary purpose of this primer preparation protocol?\",\"answer\":\"To ensure no cross-contamination between primers before they are loaded onto the automation system. It targets contamination risks across both the 1st PCR and 2nd PCR primer sets.\"},{\"question\":\"What does the protocol mean by the “one at a time” principle?\",\"answer\":\"Only one primer stock should be opened at a time, and only one primer’s aliquots should be prepared or manipulated at a time. Primer tubes are opened only once after receiving them from the vendor.\"},{\"question\":\"How are primers for the 1st PCR and 2nd PCR organized?\",\"answer\":\"The 1st PCR uses multiplexing tag primers covering rows (forward) and columns (reverse) of a 96-well plate to map samples precisely. The 2nd PCR uses universal tags (one forward and one reverse) to enable attachment to the Illumina flow cell.\"}]","Bar-Seq Library Preparation and Pooling - Primer Preparation | PDF",1789188789]