{"id":1363,"date":"2017-03-14T23:25:16","date_gmt":"2017-03-14T22:25:16","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=1363"},"modified":"2017-03-14T23:25:35","modified_gmt":"2017-03-14T22:25:35","slug":"phenolchloroformisoamyl-alcohol-extraction-and-ethanol-precipitation","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/phenolchloroformisoamyl-alcohol-extraction-and-ethanol-precipitation\/","title":{"rendered":"Phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation"},"content":{"rendered":"

We use this protocol to generate clean sterile DNA for electroporation into E. coli<\/em> or Trypanosoma brucei<\/em>.<\/p>\n

The starting\u00a0volume should be at least 100 \u03bcl.\u00a0If working with a standard ligation volume of 10 or 20\u00a0\u03bcl, make the volume up to 100\u00a0\u03bcl with distilled water in a 1.5 ml microcentrifuge tube.<\/p>\n

1. Add an equal volume of water saturated phenol:chloroform:isoamyl alcohol, vortex for 5 seconds and centrifuge at >16,000 g in a microcentrifuge for 1 minute; this separates the organic and aqueous phases.
\nSafety consideration – gloves should be worn and contact with skin should be avoided when handling phenol:chloroform. Phenol is a systemic poison whose absorption by the skin is enhanced by the presence of chlorofrm. It can cause severe burns.<\/em><\/p>\n

2. Transfer the aqueous phase (containing your DNA) to a fresh 1.5 ml microcentrifuge tube, add 0.2 \u03bcl glycogen (a ‘carrier’) and four volumes of ice cold 100% ethanol. Place at -20\u00b0C for at least 10 minutes.
\nSafety consideration – the organic phase should be transferred to your laboratory’s phenol waste and disposed of according to your institutional guidance.<\/em><\/p>\n

3. Centrifuge\u00a0at >16,000 g in a microcentrifuge for 5\u00a0minutes to pellet your DNA. Pour off the supernatant.<\/p>\n

4. Wash the pellet in 1 ml ice cold 70% ethanol, inverting several times, and centrifuge at >16,000 g in a microcentrifuge for 5\u00a0minutes. Pour off the supernatant, then\u00a0repeat the centrifugation for 30 seconds and remove the residual supernatant with a pipette.<\/p>\n

5. Open the tube and leave the pellet to dry for 5-10 minutes in a flow hood and resuspend in 10 \u03bcl distilled water.
\nIf you require your DNA to be sterile (e.g. for <\/em>T. brucei electroporation), supernatant removal\u00a0after the 70% ethanol wash, and pellet drying should be carried out in a suitable flow cabinet.<\/em><\/p>\n

 <\/p>\n","protected":false},"excerpt":{"rendered":"

We use this protocol to generate clean sterile DNA for electroporation into E. coli or…<\/p>\n","protected":false},"author":116,"featured_media":0,"parent":17,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-1363","page","type-page","status-publish","hentry","odd"],"yoast_head":"\nPhenol:chloroform:isoamyl alcohol extraction and ethanol precipitation - Alsford 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:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/phenolchloroformisoamyl-alcohol-extraction-and-ethanol-precipitation\/\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation - Alsford Lab\" \/>\n<meta property=\"og:description\" content=\"We use this protocol to generate clean sterile DNA for electroporation into E. coli or...\" \/>\n<meta property=\"og:url\" content=\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/phenolchloroformisoamyl-alcohol-extraction-and-ethanol-precipitation\/\" \/>\n<meta property=\"og:site_name\" content=\"Alsford Lab\" \/>\n<meta property=\"article:modified_time\" content=\"2017-03-14T22:25:35+00:00\" \/>\n<meta name=\"twitter:label1\" content=\"Estimated reading time\" \/>\n\t<meta name=\"twitter:data1\" content=\"1 minute\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\/\/schema.org\",\"@graph\":[{\"@type\":\"WebPage\",\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/phenolchloroformisoamyl-alcohol-extraction-and-ethanol-precipitation\/\",\"url\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/phenolchloroformisoamyl-alcohol-extraction-and-ethanol-precipitation\/\",\"name\":\"Phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation - 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