{"id":2223,"date":"2015-04-28T09:10:47","date_gmt":"2015-04-28T09:10:47","guid":{"rendered":"http:\/\/www.neb-online.fr\/?page_id=2223"},"modified":"2020-04-11T20:19:00","modified_gmt":"2020-04-11T18:19:00","slug":"chemical-transformation-tips","status":"publish","type":"page","link":"https:\/\/www.neb-online.fr\/en\/competent-cells\/chemical-transformation-tips\/","title":{"rendered":"Chemical Transformation Tips"},"content":{"rendered":"<div class=\"wpb-content-wrapper\"><p>[vc_row][vc_column][vc_column_text]<\/p>\n<h3>Thawing<\/h3>\n<ul>\n<li>Cells are best thawed on ice.<\/li>\n<li>DNA should be added as soon as the last trace of ice in the tube disappears.<\/li>\n<li>Cells can be thawed by hand, but warming above 0\u00b0C decreases efficiency.<\/li>\n<\/ul>\n<h3>DNA Quality<\/h3>\n<ul>\n<li>DNA for transformation should be purified and resuspended in water or TE Buffer.<\/li>\n<li>Up to 10 \u00b5l of DNA from a ligation mix can be used with only a 2-fold loss of efficiency.<\/li>\n<li>To maximize transformants, purification by either a spin column or phenol\/chloroform extraction and ethanol precipitation should be performed.<\/li>\n<\/ul>\n<p>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=&#8221;1\/2&#8243;][vc_column_text]<\/p>\n<h3>DNA\u00a0Amount<\/h3>\n<p>The optimal amount of DNA is lower than commonly recognized. Using clean, supercoiled pUC19, the efficiency of transformation is highest in the 100 pg-1 ng range. However, the total colonies which can be obtained from a single transformation reaction increase up to about 100 ng.[\/vc_column_text][\/vc_column][vc_column width=&#8221;1\/2&#8243;][vc_single_image image=&#8221;1102&#8243; img_size=&#8221;full&#8221;][vc_column_text css=&#8221;.vc_custom_1431961302808{margin-top: -25px !important;}&#8221;]<span class=\"bildunterschrift\"><em>DNA Effects on Transformation Efficiency and Colony Output: The optimal amount of DNA to use in a transformation reaction is lower than commonly recognized. Using clean, supercoiled pUC19, the efficiency of transformation is highest in the 100 pg-1 ng range. However, the total colonies which can be obtained from a single transformation reaction increase up to about 100 ng.<\/em><\/span>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=&#8221;1\/2&#8243;][vc_column_text]<\/p>\n<h3>Incubation of DNA with Cells on Ice<\/h3>\n<p>Incubate on ice for 30 minutes. Expect a 2-fold loss in TE for every 10 minutes you shorten this step.[\/vc_column_text][\/vc_column][vc_column width=&#8221;1\/2&#8243;][vc_single_image image=&#8221;1105&#8243; img_size=&#8221;full&#8221;][vc_column_text css=&#8221;.vc_custom_1431961307913{margin-top: -25px !important;}&#8221;]<span class=\"bildunterschrift\"><em>Effect of DNA incubation time on NEB 5-alpha competent E.coli transformation efficiency: 50 \u03bcl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except DNA incubation time varied from 0 to 40 minutes.<\/em><\/span>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=&#8221;1\/2&#8243;][vc_column_text]<\/p>\n<h3>Heat Shock<\/h3>\n<p>Both temperature and time are specific to the transformation volume and vessel. Typically, 30 seconds at 42\u00b0C is recommended, except when using BL21 (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/C2530@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #C2530<\/a>) which requires exactly 10 seconds.[\/vc_column_text][\/vc_column][vc_column width=&#8221;1\/2&#8243;][vc_single_image image=&#8221;1104&#8243; img_size=&#8221;full&#8221;][vc_column_text css=&#8221;.vc_custom_1431961313497{margin-top: -25px !important;}&#8221;]<span class=\"bildunterschrift\"><em>Effect of heat shock time on NEB 5-alpha competent E.coli transformation efficiency: 50 \u03bcl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except heat shock time varied from 0 to 80 seconds.<\/em><\/span>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=&#8221;1\/2&#8243;][vc_column_text]<\/p>\n<h3>Outgrowth<\/h3>\n<ul>\n<li>Outgrowth at 37\u00b0C for 1 hour is best for cell recovery and for expression of antibiotic resistance.<br \/>\nExpect a 2-fold loss in TE for every 15 minutes you shorten this step.<\/li>\n<li>SOC gives 2-fold higher TE than LB medium.<\/li>\n<li>Incubation with shaking or rotating the tube gives 2-fold higher TE.<\/li>\n<\/ul>\n<p>[\/vc_column_text][\/vc_column][vc_column width=&#8221;1\/2&#8243;][vc_single_image image=&#8221;1106&#8243; img_size=&#8221;full&#8221;][vc_column_text css=&#8221;.vc_custom_1431961319361{margin-top: -25px !important;}&#8221;]<span class=\"bildunterschrift\"><em>Effect of outgrowth medium on transformation efficiency: 50 \u03bcl of NEB 5-alpha competent E.coli was transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol with the exception of varying the outgrowth medium. NEB SOC outgrowth medium delivers the highest transformation efficiency.<\/em><\/span>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n<h3>Plating<\/h3>\n<ul>\n<li>Selection plates can be used warm or cold, wet or dry with no significant effects on TE.<\/li>\n<li>Warm, dry plates are easier to spread and allow for the most rapid colony formation.<\/li>\n<\/ul>\n<p>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=&#8221;1\/2&#8243;][vc_single_image image=&#8221;1103&#8243; img_size=&#8221;full&#8221;][vc_column_text css=&#8221;.vc_custom_1431961324451{margin-top: -25px !important;}&#8221;]<span class=\"bildunterschrift\"><em>Benefit from the high transformation efficiencies of NEB 5-alpha: The transformation efficiencies of NEB 5-alpha and DH5\u03b1 were compared using each manufacturer&#8217;s recommended protocols. Values shown are the average of triplicate experiments.<\/em><\/span>[\/vc_column_text][\/vc_column][vc_column width=&#8221;1\/2&#8243;][vc_single_image image=&#8221;1107&#8243; img_size=&#8221;full&#8221;][vc_column_text css=&#8221;.vc_custom_1431961329485{margin-top: -25px !important;}&#8221;]<span class=\"bildunterschrift\"><em>Take advantage of the low cost per transformation with NEB 5-alpha. Calculations were based on list price and recommended transformation volumes.<\/em><\/span>[\/vc_column_text][\/vc_column][\/vc_row]<\/p>\n<\/div>","protected":false},"excerpt":{"rendered":"<p>[vc_row][vc_column][vc_column_text] Thawing Cells are best thawed on ice. DNA should be added as soon as the last trace of ice in the tube disappears. Cells can be thawed by hand, but warming above 0\u00b0C decreases efficiency. DNA Quality DNA for transformation should be purified and resuspended in water or TE Buffer. Up to 10 \u00b5l&#8230;  <a class=\"excerpt-read-more\" href=\"https:\/\/www.neb-online.fr\/en\/competent-cells\/chemical-transformation-tips\/\" title=\"Read Chemical Transformation Tips\">Read more &raquo;<\/a><\/p>\n","protected":false},"author":15,"featured_media":9893,"parent":2206,"menu_order":0,"comment_status":"open","ping_status":"open","template":"page-sidebar-competent-cells.php","meta":{"footnotes":""},"class_list":["post-2223","page","type-page","status-publish","has-post-thumbnail","hentry"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.0 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Chemical Transformation Tips - New England Biolabs France<\/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.neb-online.fr\/en\/competent-cells\/chemical-transformation-tips\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Chemical Transformation Tips - New England Biolabs France\" \/>\n<meta property=\"og:description\" content=\"[vc_row][vc_column][vc_column_text] Thawing Cells are best thawed on ice. 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