{"id":2300,"date":"2017-06-08T20:53:52","date_gmt":"2017-06-09T00:53:52","guid":{"rendered":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/"},"modified":"2018-07-20T16:40:40","modified_gmt":"2018-07-20T20:40:40","slug":"plasmid-isolation","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/","title":{"rendered":"Plasmid Isolation from Yeast using Qiagen “QiaPrep” Kit"},"content":{"rendered":"
\n

A Dohlman Lab Protocol<\/em><\/p>\n

\n

1. Grow culture to saturation in 4 ml selective media<\/p>\n

2. Spin 1.5 ml for 10 min in microfuge tube, and remove supe<\/p>\n

3. Add another 1.5ml of culture to pellet, spin, and remove supe (optional)<\/p>\n

4. Resuspend in 500 ul sterile dd H2O<\/p>\n

5. Spin 10 min, remove supe<\/p>\n

6. Resuspend pellet in 250 ul Qiagen buffer P1 (containing RNAse A)<\/p>\n

7. Add 250 ul Qiagen buffer P2 and 250 ul acid washed glass beads<\/p>\n

8. Vortex for 2 min. Let sit 5 min in P2. Do this at 4C.<\/p>\n

9. Add 350 ul chilled Qiagen buffer N3<\/p>\n

10. Mix by inverting, then incubate on ice for 5 min.<\/p>\n

11. Spin 10 min<\/p>\n

12. Place Qiaprep \u2013 spin column in 2 ml microfuge tube and apply supe (after the spin)<\/p>\n

13. Spin 30-60 sec, drain tube<\/p>\n

14. Wash column with 0.5 ml Qiagen buffer PB<\/p>\n

15. Spin 30-60 sec, drain tube<\/p>\n

16. Wash column with 0.75 ml Qiagen buffer PE<\/p>\n

17. Spin 30-60 sec, drain tube, spin again<\/p>\n

18. Place column in clean 1.5 ml microfuge tube<\/p>\n

19. Elute DNA with 50-100 ul TE (10 mM Tris-HCl pH 8.5, 1 mM EDTA) or dd H2O<\/p>\n

20. Spin 30 sec<\/p>\n

21. Transform a range of plasmid amounts into E. coli to amplify<\/p>\n

ALTERNATIVE METHOD: We have started using the Zymoprep Yeast Plasmid Miniprep Kit II<\/a> with much success.\u00a0 Make sure you select Miniprep II as the kit.<\/p>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

A Dohlman Lab Protocol 1. Grow culture to saturation in 4 ml selective media 2. Spin 1.5 ml for 10 min in microfuge tube, and remove supe 3. Add another 1.5ml of culture to pellet, spin, and remove supe (optional) 4. Resuspend in 500 ul sterile dd H2O 5. Spin 10 min, remove supe 6. … Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":13,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2300","page","type-page","status-publish","hentry","odd"],"acf":[],"yoast_head":"\nPlasmid Isolation from Yeast using Qiagen "QiaPrep" Kit - Dohlman Lab<\/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.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Plasmid Isolation from Yeast using Qiagen "QiaPrep" Kit - Dohlman Lab\" \/>\n<meta property=\"og:description\" content=\"A Dohlman Lab Protocol 1. Grow culture to saturation in 4 ml selective media 2. Spin 1.5 ml for 10 min in microfuge tube, and remove supe 3. Add another 1.5ml of culture to pellet, spin, and remove supe (optional) 4. Resuspend in 500 ul sterile dd H2O 5. Spin 10 min, remove supe 6. … Read more\" \/>\n<meta property=\"og:url\" content=\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/\" \/>\n<meta property=\"og:site_name\" content=\"Dohlman Lab\" \/>\n<meta property=\"article:modified_time\" content=\"2018-07-20T20:40:40+00:00\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data1\" content=\"1 minute\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\/\/schema.org\",\"@graph\":[{\"@type\":\"WebPage\",\"@id\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/\",\"url\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/\",\"name\":\"Plasmid Isolation from Yeast using Qiagen \\\"QiaPrep\\\" Kit - Dohlman Lab\",\"isPartOf\":{\"@id\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/#website\"},\"datePublished\":\"2017-06-09T00:53:52+00:00\",\"dateModified\":\"2018-07-20T20:40:40+00:00\",\"breadcrumb\":{\"@id\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/#breadcrumb\"},\"inLanguage\":\"en-US\",\"potentialAction\":[{\"@type\":\"ReadAction\",\"target\":[\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/\"]}]},{\"@type\":\"BreadcrumbList\",\"@id\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/#breadcrumb\",\"itemListElement\":[{\"@type\":\"ListItem\",\"position\":1,\"name\":\"Home\",\"item\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/\"},{\"@type\":\"ListItem\",\"position\":2,\"name\":\"Resources\",\"item\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/\"},{\"@type\":\"ListItem\",\"position\":3,\"name\":\"Laboratory Methods\",\"item\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/\"},{\"@type\":\"ListItem\",\"position\":4,\"name\":\"Plasmid Isolation from Yeast using Qiagen “QiaPrep” Kit\"}]},{\"@type\":\"WebSite\",\"@id\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/#website\",\"url\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/\",\"name\":\"Dohlman Lab\",\"description\":\"Department of Pharmacology\",\"potentialAction\":[{\"@type\":\"SearchAction\",\"target\":{\"@type\":\"EntryPoint\",\"urlTemplate\":\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/?s={search_term_string}\"},\"query-input\":{\"@type\":\"PropertyValueSpecification\",\"valueRequired\":true,\"valueName\":\"search_term_string\"}}],\"inLanguage\":\"en-US\"}]}<\/script>\n<!-- \/ Yoast SEO plugin. -->","yoast_head_json":{"title":"Plasmid Isolation from Yeast using Qiagen \"QiaPrep\" Kit - Dohlman Lab","robots":{"index":"index","follow":"follow","max-snippet":"max-snippet:-1","max-image-preview":"max-image-preview:large","max-video-preview":"max-video-preview:-1"},"canonical":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/","og_locale":"en_US","og_type":"article","og_title":"Plasmid Isolation from Yeast using Qiagen \"QiaPrep\" Kit - Dohlman Lab","og_description":"A Dohlman Lab Protocol 1. Grow culture to saturation in 4 ml selective media 2. Spin 1.5 ml for 10 min in microfuge tube, and remove supe 3. Add another 1.5ml of culture to pellet, spin, and remove supe (optional) 4. Resuspend in 500 ul sterile dd H2O 5. Spin 10 min, remove supe 6. … Read more","og_url":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/","og_site_name":"Dohlman Lab","article_modified_time":"2018-07-20T20:40:40+00:00","twitter_card":"summary_large_image","twitter_misc":{"Est. reading time":"1 minute"},"schema":{"@context":"https:\/\/schema.org","@graph":[{"@type":"WebPage","@id":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/","url":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/","name":"Plasmid Isolation from Yeast using Qiagen \"QiaPrep\" Kit - Dohlman Lab","isPartOf":{"@id":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/#website"},"datePublished":"2017-06-09T00:53:52+00:00","dateModified":"2018-07-20T20:40:40+00:00","breadcrumb":{"@id":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/#breadcrumb"},"inLanguage":"en-US","potentialAction":[{"@type":"ReadAction","target":["https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/"]}]},{"@type":"BreadcrumbList","@id":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/plasmid-isolation\/#breadcrumb","itemListElement":[{"@type":"ListItem","position":1,"name":"Home","item":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/"},{"@type":"ListItem","position":2,"name":"Resources","item":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/"},{"@type":"ListItem","position":3,"name":"Laboratory Methods","item":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/"},{"@type":"ListItem","position":4,"name":"Plasmid Isolation from Yeast using Qiagen “QiaPrep” Kit"}]},{"@type":"WebSite","@id":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/#website","url":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/","name":"Dohlman Lab","description":"Department of Pharmacology","potentialAction":[{"@type":"SearchAction","target":{"@type":"EntryPoint","urlTemplate":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/?s={search_term_string}"},"query-input":{"@type":"PropertyValueSpecification","valueRequired":true,"valueName":"search_term_string"}}],"inLanguage":"en-US"}]}},"_links_to":[],"_links_to_target":[],"_links":{"self":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2300","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/users\/22429"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/comments?post=2300"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2300\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2239"}],"wp:attachment":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/media?parent=2300"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}