{"id":294,"date":"2023-07-11T13:18:36","date_gmt":"2023-07-11T20:18:36","guid":{"rendered":"https:\/\/live-usc-dornsife.pantheonsite.io\/pombenet\/?page_id=294"},"modified":"2023-08-03T13:28:32","modified_gmt":"2023-08-03T20:28:32","slug":"hydroxylamine-mutagenesis","status":"publish","type":"page","link":"https:\/\/dornsife.usc.edu\/pombenet\/hydroxylamine-mutagenesis\/","title":{"rendered":"Hydroxylamine Mutagenesis of plasmid DNA"},"content":{"rendered":"\n\n  \n    \n\n\n\n\n\n\n<div\n  class=\"cc--component-container cc--rich-text \"\n\n  \n  \n  \n  \n  \n  \n  >\n  <div class=\"c--component c--rich-text\"\n    \n      >\n\n    \n      \n<div class=\"f--field f--wysiwyg\">\n\n    \n  <div class=\"article-list\">\n<div class=\"article cf\">\n<div class=\"html-content\">\n<p>We use this method for random mutagenesis of plasmid DNA which is then used in a\u00a0<a class=\" RLC_Start RLC_Ok\" href=\"\/pombenet\/working-with-pombe\/\">plasmid shuffle<\/a>\u00a0or screen for\u00a0<a class=\" RLC_Start RLC_Ok\" href=\"\/pombenet\/working-with-pombe\/\">ts mutants<\/a>. It is derived from the protocol in Rose and Fink,\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=3030557&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/3030557\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=3030557&amp;dopt=Abstract\">Cell 48:1047-60 (1987)\u00a0<\/a>.<\/p>\n<p>&nbsp;<\/p>\n<\/div>\n<\/div>\n<div class=\"article cf\">\n<h3 class=\"article-title\">1. Prepare 1M Hydroxylamine solution.<\/h3>\n<div class=\"html-content\">\n<ul>\n<li>0.35g hydroxylamine hydrochloride (H3NO-HCl)<\/li>\n<li>450 \u00b5l 5M NaOH<\/li>\n<li>4.55 ml ice cold sterile MQ water<\/li>\n<\/ul>\n<p>Make sure pH is around 6.7 and keep solution on ice<\/p>\n<p>&nbsp;<\/p>\n<\/div>\n<\/div>\n<div class=\"article cf\">\n<h3 class=\"article-title\">2. Treatment<\/h3>\n<div class=\"html-content\">\n<p>Incubate 10 \u00b5g plasmid DNA with 500 \u00b5l hydroxylamine solution for 20 h at 37 \u00b0C in an eppendorf tube. Seal eppendorf tube with parafilm.<\/p>\n<p>&nbsp;<\/p>\n<\/div>\n<\/div>\n<div class=\"article cf\">\n<h3 class=\"article-title\">3. Purification<\/h3>\n<div class=\"html-content\">\n<p>Purify DNA from hydroxylamine using Qiagen column&#8230;(Purification of plasmid DNA prepared by other methods&#8230;page 31 of 7\/99 QIAprep Miniprep Handbood)<\/p>\n<ul>\n<li>add five volumes of Buffer PB to one volume of DNA and mix<\/li>\n<li>load column (0.75 mls at a time) and centrifuge 1 min at max speed<\/li>\n<li>wash column with 0.75 mls Buffer PE (stand 1 min, centrifuge 1 min at max speed)<\/li>\n<li>centrifuge one more time (1 -3 minutes at max speed)<\/li>\n<li>elute DNA with TE or EB (50 \u00b5l), stand 1 min, centrifuge into new tube)<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<\/div>\n<\/div>\n<div class=\"article cf\">\n<h3 class=\"article-title\">4. Recovery<\/h3>\n<div class=\"html-content\">\n<p>Check recovery of DNA from mutagenesis by running on agarose gel and transform into appropriate yeast strain. Use approximately 500 ng to 1 \u00b5g DNA per transformation.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Forsburg Lab anecdotes:<\/strong><br \/>\nWe used a plasmid shuffle strategy for\u00a0<i>mcm7<\/i>. We mutagenized from 16 to 22.5 h at 36 *C or 1 h at 70 *C, transforming by electroporation with 5 \u00b5l mutagenized DNA per electroporation. We screened about 10,000 transformants and recovered 3 ts mutants (all mutagenized for 22.5 h at 36*C). Two contained identical mutations. One of the mutants did not support growth when reintegrated into the genome in single copy. The other is a well behaved, tight ts.<\/p>\n<\/div>\n<\/div>\n<\/div>\n\n\n\n<\/div>\n\n\n  <\/div><\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":354,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-content-detail.php","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-294","page","type-page","status-publish","hentry"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.1.1 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Hydroxylamine Mutagenesis of plasmid DNA - PombeNet Forsburg Lab<\/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:\/\/dornsife.usc.edu\/pombenet\/hydroxylamine-mutagenesis\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Hydroxylamine Mutagenesis of plasmid DNA - 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