{"id":2070,"date":"2021-07-30T11:09:43","date_gmt":"2021-07-30T11:09:43","guid":{"rendered":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/?page_id=2070"},"modified":"2021-07-30T11:26:11","modified_gmt":"2021-07-30T11:26:11","slug":"preparation-of-electrocompetent-e-coli-cells","status":"publish","type":"page","link":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/protocols\/preparation-of-electrocompetent-e-coli-cells\/","title":{"rendered":"Preparation of electrocompetent E. coli cells"},"content":{"rendered":"\n<div class=\"vf-grid | vf-grid__col-3\"><div class=\"vf-grid__col--span-2\"><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<h3 class=\"wp-block-heading\">Before starting:<\/h3>\n\n\n\n<ul class=\"wp-block-list\"><li>Prepare all required reagents (SOB medium, sterile 10% glycerol)<\/li><li>Make sure you have access to a pre-cooled centrifuge (4\u00b0C) and liquid nitrogen<\/li><li>Prepare sterile, pre-cooled centrifuge tubes (4\u00b0C) and 1.5 ml Eppendorf tubes<\/li><\/ul>\n\n\n\n<h3 class=\"wp-block-heading\">Protocol:<\/h3>\n\n\n\n<ol class=\"wp-block-list\"><li>Inoculate 30 mL SOB with a single colony from a fresh plate and grow the culture overnight at 37\u00b0C<\/li><li>Inoculate 1 L SOB with 10 ml of the preculture (use a ratio of 1:100 for the inoculation) and grow it at 37\u00b0C, 200 rpm until the OD<sub>600<\/sub> ~ 0.4-0.5 (takes approximately 4 h); continue working on ice or at 4\u00b0C for all steps from here on!<\/li><li>Centrifuge the cells at 4000 rpm \/ 20 min \/ 4\u00b0C<\/li><li>Wash the cell pellet with 1 volume of sterile 10 % glycerol<\/li><li>Centrifuge the cells at 4000 rpm \/ 20 min \/ 4\u00b0C<\/li><li>Wash the cell pellet with 0.5 volumes of sterile 10 % glycerol<\/li><li>Centrifuge the cells at 4000 rpm \/ 20 min \/ 4\u00b0C<\/li><li>Wash the cell pellet with 0.1 volumes of sterile 10 % glycerol<\/li><li>Centrifuge the cells at 4000 rpm \/ 20 min \/ 4\u00b0C<\/li><li>Add 0.5 &#8211; 1 mL of sterile 10 % glycerol (per liter of liquid culture you started with); resuspend the cell pellet<\/li><li>Prepare 100 \u00b5l aliquots in 1.5 mL Eppendorf tubes and freeze them immediately in liquid nitrogen<\/li><li>Store the electrocompetent cells at -80\u00b0C<\/li><\/ol>\n\n\n\n<h4 class=\"wp-block-heading\">SOB medium (per liter):<\/h4>\n\n\n\n<ul class=\"wp-block-list\"><li>20 g Bacto Tryptone<\/li><li>5 g Bacto Yeast Extract<\/li><li>8.6 mM NaCl<\/li><li>2.5 mM KCl<\/li><\/ul>\n\n\n\n<ul class=\"wp-block-list\"><li>pH 7.0<\/li><li>auto-clave<\/li><li>add sterile 10 mM MgCl<sub>2<\/sub> and 10 mM MgSO<sub>4<\/sub><\/li><\/ul>\n\n<\/div>\n<\/div>\n\n\n<div><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":0,"parent":2060,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"template-title-left-aligned.php","meta":{"_acf_changed":false,"footnotes":""},"embl_taxonomy":[],"class_list":["post-2070","page","type-page","status-publish","hentry"],"acf":[],"embl_taxonomy_terms":[],"_links":{"self":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2070","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/comments?post=2070"}],"version-history":[{"count":3,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2070\/revisions"}],"predecessor-version":[{"id":2132,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2070\/revisions\/2132"}],"up":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2060"}],"wp:attachment":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/media?parent=2070"}],"wp:term":[{"taxonomy":"embl_taxonomy","embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/embl_taxonomy?post=2070"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}