{"id":2072,"date":"2021-07-30T11:09:47","date_gmt":"2021-07-30T11:09:47","guid":{"rendered":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/?page_id=2072"},"modified":"2021-07-30T11:25:57","modified_gmt":"2021-07-30T11:25:57","slug":"preparation-of-chemically-competent-e-coli-cells","status":"publish","type":"page","link":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/protocols\/preparation-of-chemically-competent-e-coli-cells\/","title":{"rendered":"Preparation of chemically competent 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 and solutions<\/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>Streak out the desired bacterial cell line on an LB-agar plate and incubate overnight at 37\u00b0C<\/li><li>Inoculate 250 ml SOB and grow at 18-20\u00b0C, 200 rpm until the OD<sub>600<\/sub> ~ 0.4-0.6 (takes approximately 24-36 h); continue working on ice or at 4\u00b0C for all steps from here on!<\/li><li>Place the flask on ice for 10 min<\/li><li>Centrifuge the cells at 4000 rpm \/ 10 min \/ 4\u00b0C<\/li><li>Gently resuspend the cells in 80 ml ice-cold TB and keep on ice for 10 min.<\/li><li>Centrifuge the cells at 4000 rpm \/ 10 min \/ 4\u00b0C<\/li><li>Gently resuspend the cells in 20 ml ice-cold TB and 1.4 ml DMSO and keep on ice for 10 min.<\/li><li>Prepare 50 \u00b5l aliquots in 1.5 mL Eppendorf tubes and freeze them immediately in liquid nitrogen<\/li><li>Store the chemically competent 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\n\n<h4 class=\"wp-block-heading\">TB solution<\/h4>\n\n\n\n<ul class=\"wp-block-list\"><li>10 mM PIPES<\/li><li>250 mM KCl<\/li><li>15 mM CaCl<sub>2<\/sub><\/li><\/ul>\n\n\n\n<ul class=\"wp-block-list\"><li>Adjust to pH 6.7 with KOH<\/li><li>Add 55 mM MnCl<sub>2<\/sub><\/li><li>Sterilize by filtration (0.45 \u00b5m filter) and store at 4\u00b0C<\/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-2072","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\/2072","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=2072"}],"version-history":[{"count":3,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2072\/revisions"}],"predecessor-version":[{"id":2130,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2072\/revisions\/2130"}],"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=2072"}],"wp:term":[{"taxonomy":"embl_taxonomy","embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/embl_taxonomy?post=2072"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}