{"id":2296,"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\/lazybones\/"},"modified":"2018-07-20T16:35:07","modified_gmt":"2018-07-20T20:35:07","slug":"lazybones","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/lazybones\/","title":{"rendered":"PLATE Transformation"},"content":{"rendered":"<div>\n<p><em>A Dohlman Lab Protocol<\/em><\/p>\n<div>\n<p>\u201cPLATE\u201d is an acronym of the names of ingredients in the transformation solution: PEG, Lithium Acetate, Tris, and EDTA.<\/p>\n<p>-Use sterile technique and sterile solutions throughout this method.-<\/p>\n<p>1. In a 15 ml culture tube, inoculate 2-3 ml of the appropriate media with a single colony of the yeast strain to be transformed. This is the starter culture.<\/p>\n<ul>\n<li>Various wild-type and mutant yeast strains can be obtained from Research Genetics (Huntsville, AL) or ATCC (Manassas, VA).<\/li>\n<\/ul>\n<p>2. Grow the starter culture at 30 with shaking (250 rpm) until it reaches saturation.<\/p>\n<ul>\n<li>This takes anywhere from 1 to 6 days, depending on the strain and the media.<\/li>\n<\/ul>\n<p>3. Place 0.5 ml of the saturated culture in a sterile microfuge tube.<\/p>\n<p>4. Collect the cells by centrifuging at 16,000 x g for 30 sec.<\/p>\n<p>5. Aspirate the supernatant.<\/p>\n<p>6. Add 10 ul of sonicated salmon sperm DNA (10 mg\/ml stock) (Stratagene, #201190).<\/p>\n<ul>\n<li>The DNA must be single-stranded, which can be achieved by boiling for 5 min (a 100\u00b0C heat block works well) and immediately chilling on ice.<\/li>\n<li>The DNA only needs to be boiled every 3 to 4 times it is used (as long as it remains on ice when thawed).<\/li>\n<\/ul>\n<p>7. Add 1-2 ug of the plasmid DNA to be transformed and vortex.<\/p>\n<p>8. Add 500 ul of PLATE solution. Mix by inverting or by pipetting gently. Do not vortex.<\/p>\n<p><strong>PLATE solution<\/strong><br \/>\n40% PEG3350 (w\/v)<br \/>\n100 mM lithium acetate (LiAc)<br \/>\n10 mM Tris, pH 7.5<br \/>\n0.4 mM EDTA<\/p>\n<p>9. Leave at room temperature or 30oC for 15 min, then heat shock 42oC for 15 min, then ice for 2 min. Alternatively leave at room temperature or at 30oC for 24-48 hours.<\/p>\n<p>10. Collect the cells by centrifuging at 16,000 x g for 30 sec.<\/p>\n<p>11. Aspirate the supernatant.<\/p>\n<p>12. Resuspend the cells in 200 ul of sterile water by pipetting gently and thoroughly.<\/p>\n<p>13. Spread on solid media that will select for the plasmid.<\/p>\n<p>14. Incubate at 30\u00b0 until colonies appear.<\/p>\n<ul>\n<li>This takes around 2 to 6 days, depending on the yeast strain and the plasmid\u2019s nutritional marker. In our experience, it usually takes ~ 2 days for LEU2 plasmids and ~ 6 days for URA3 plasmids.<\/li>\n<\/ul>\n<p>15. Restreak 2-3 transformed colonies onto a new plate.<\/p>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>A Dohlman Lab Protocol \u201cPLATE\u201d is an acronym of the names of ingredients in the transformation solution: PEG, Lithium Acetate, Tris, and EDTA. -Use sterile technique and sterile solutions throughout this method.- 1. In a 15 ml culture tube, inoculate 2-3 ml of the appropriate media with a single colony of the yeast strain to &hellip; <a href=\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/lazybones\/\" aria-label=\"Read more about PLATE Transformation\">Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":9,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2296","page","type-page","status-publish","hentry","odd"],"acf":[],"_links_to":[],"_links_to_target":[],"_links":{"self":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2296","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=2296"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2296\/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=2296"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}