{"id":462,"date":"2016-11-09T15:36:46","date_gmt":"2016-11-09T23:36:46","guid":{"rendered":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/?page_id=462"},"modified":"2023-04-27T12:39:18","modified_gmt":"2023-04-27T19:39:18","slug":"protocol-3-e-coli-transformation","status":"publish","type":"page","link":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/protocol-3-e-coli-transformation\/","title":{"rendered":"Protocol 3: E.coli transformation"},"content":{"rendered":"\n<div class=\"wp-block-file\"><a id=\"wp-block-file--media-5864b16b-c918-4988-810b-2dc4ae66d650\" href=\"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-content\/uploads\/2023\/04\/PROTOCOL3-Ecoli-Transformation.doc\">PROTOCOL3-Ecoli-Transformation<\/a><a href=\"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-content\/uploads\/2023\/04\/PROTOCOL3-Ecoli-Transformation.doc\" class=\"wp-block-file__button wp-element-button\" download aria-describedby=\"wp-block-file--media-5864b16b-c918-4988-810b-2dc4ae66d650\">Download<\/a><\/div>\n\n\n\n<p><strong><em>E. coli<\/em><\/strong><strong>&nbsp;transformation<\/strong><\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp;&nbsp; 1. Thaw frozen (-80 \u00b0 C) competent cells on ice<\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp; 2. Mix 1 \u00b5l DNA <\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp;&nbsp;3. Incubate on ice 20 min<\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp; 4. Incubate <strong>42 \u00b0<\/strong> C 45 sec-1 min<\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp; 5. Incubate on ice 2 min<\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp; 6. Add 1 ml LB <\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp;&nbsp;7. Incubate at 37 \u00b0 C 45 min-1hour<\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp; 8. Spin cells down (2min at 14000rpm) <\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp;&nbsp;9.&nbsp; Remove supernatant,&nbsp; keep cell pellet in a small amount of medium &nbsp;(about 50-100 \u00b5l. <\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;&nbsp;&nbsp;10. Re-suspend cells in the medium, transfer everything on LB agar plate&nbsp; (+antibiotic) and spread cells with a blue sterile spreader. &nbsp;(The goal is to evenly distribute cells&nbsp; and to allow them to be absorbed into the agar). <\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">&nbsp;<\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\"><strong>LB ((+antibiotic) agar plate:<\/strong><\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">1.&nbsp;&nbsp;&nbsp; Heat 150 ml LA agar in a microwave oven (9 \u2013for 3 min on old microwave or power 5 for 3 min on new) until completely melted. <\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">2.&nbsp;&nbsp;&nbsp; After cooling the solution and add antibiotic &nbsp;<\/pre>\n\n\n\n<pre class=\"wp-block-preformatted\">3.&nbsp;&nbsp;&nbsp; Make Petri dishes (150 ml LA makes 5-6 plates)<\/pre>\n","protected":false},"excerpt":{"rendered":"<p>E. coli&nbsp;transformation &nbsp;&nbsp;&nbsp; 1. Thaw frozen (-80 \u00b0 C) competent cells on ice &nbsp;&nbsp; 2. Mix 1 \u00b5l DNA &nbsp;&nbsp;&nbsp;3. Incubate on ice 20 min &nbsp;&nbsp; 4. Incubate 42 \u00b0 C 45 sec-1 min &nbsp;&nbsp; 5. Incubate on ice 2 min &nbsp;&nbsp; 6. Add 1 ml LB &nbsp;&nbsp;&nbsp;7. Incubate at 37 \u00b0 C 45 min-1hour [&hellip;]<\/p>\n","protected":false},"author":4,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"off","_et_pb_old_content":"","_et_gb_content_width":"","footnotes":""},"class_list":["post-462","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/pages\/462","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/users\/4"}],"replies":[{"embeddable":true,"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/comments?post=462"}],"version-history":[{"count":3,"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/pages\/462\/revisions"}],"predecessor-version":[{"id":1416,"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/pages\/462\/revisions\/1416"}],"wp:attachment":[{"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/media?parent=462"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}