{"id":873,"date":"2013-09-13T15:46:36","date_gmt":"2013-09-13T14:46:36","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=873"},"modified":"2014-11-26T11:30:03","modified_gmt":"2014-11-26T10:30:03","slug":"electroporation","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/electroporation\/","title":{"rendered":"Electroporation"},"content":{"rendered":"

We use two protocols to transfect linearised DNA constructs into bloodstream form T. brucei<\/em>, depending on the integration target and the desired transformation efficiency – both rely on a nucleofection apparatus running progamme X-001 (Lonza<\/a>)<\/p>\n

Protocol 1: standard<\/strong><\/em><\/p>\n

This is particularly useful for transfecting pRPa constructs, which integrate at the ‘landing pad locus’ in 2T1<\/a> cells, or similarly efficient construct-target combinations<\/p>\n

    \n
  1. Digest 10 \u00b5g construct with the appropriate restriction enzyme(s) to generate a linear DNA molecule with termini to allow integration into the T. brucei<\/em> genome by homologous recombination<\/li>\n
  2. Clean the digested construct by phenol:chloroform:isoamylalcohol extraction and ethanol precipitation (do not<\/strong> include any salts such as sodium acetate, as these will interfere with electroporation); resuspend the precipitated DNA in 10 \u00b5l sterile distilled water<\/li>\n
  3. Pellet 25 million bloodstream form T. brucei<\/em> at 1000 g for 10 minutes<\/li>\n
  4. Pour off the supernatant, remove as much of the residual media as possible without disturbing the cell pellet, and resuspend in 100 \u00b5l cytomix (pre-warmed to 37\u00b0C) plus 10 \u00b5l DNA solution<\/li>\n
  5. Transfer cell-cytomix-DNA mixture to a 2 mm gap cuvette and apply nucleofection programme X-001<\/li>\n
  6. Transfer cells to 60 ml media and add appropriate selective drugs 4-6 hours later<\/li>\n
  7. Transfer to two 48 well plates: ‘neat’ and ‘5-fold diluted’; positive wells should be transferred to 10 ml cultures five days post-electroporation<\/li>\n<\/ol>\n

    Cytomix<\/strong> (filter sterilise and store at 4\u00b0C)<\/p>\n\n\n\n\n\n\n\n\n\n\n\n
    KCl<\/span><\/td>\n120 mM<\/span><\/td>\n<\/tr>\n
    HEPES pH7.6<\/span><\/td>\n25 mM<\/span><\/td>\n<\/tr>\n
    1<\/sup>K2<\/sub>HPO4<\/sub>\/KH2<\/sub>PO4<\/sub> pH7.6<\/span><\/td>\n10 mM<\/span><\/td>\n<\/tr>\n
    MgCl2<\/sub>.6H2<\/sub>O<\/span><\/td>\n5 mM<\/span><\/td>\n<\/tr>\n
    EGTA pH7.6<\/span><\/td>\n2 mM<\/span><\/td>\n<\/tr>\n
    CaCl2<\/sub><\/span><\/td>\n150 \u00b5M<\/span><\/td>\n<\/tr>\n
    Glucose<\/span><\/td>\n0.5%<\/span><\/td>\n<\/tr>\n
    Albumin, bovine serum<\/span><\/td>\n100 \u00b5g\/ml<\/span><\/td>\n<\/tr>\n
    2<\/sup>Hypoxanthine<\/span><\/td>\n1 mM<\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

    1<\/sup> 10x K2<\/sub>HPO4<\/sub>\/KH2<\/sub>PO4<\/sub> pH7.6: 8.66ml 1 M K2<\/sub>HPO4<\/sub>, 1.34 ml 1M KH2<\/sub>PO4<\/sub>, 90 ml H2<\/sub>O (store at room temperature)
    \n2<\/sup> 100x Hypoxanthine: 0.4 g NaOH, 100 ml H2<\/sub>O, 1.36 g hypoxanthine (store at -20\u00b0C)<\/p>\n

    Recommended drug concentrations<\/strong><\/p>\n\n\n\n\n\n\n\n\n
    Drug<\/span><\/strong><\/td>\nStock (mg\/ml)<\/strong>
    \n<\/span><\/td>\n    		Selective (\u00b5g\/ml)<\/strong>
    \n<\/span><\/td>\n    		Maintenance (\u00b5g\/ml)<\/strong>
    \n<\/span><\/td>\n<\/tr>\n
    Phleomycin<\/span><\/em><\/td>\n10 mg\/ml<\/span><\/td>\n2<\/span><\/td>\n1
    \n<\/span><\/td>\n<\/tr>\n
    G418<\/span><\/em><\/td>\n10 mg\/ml<\/span><\/td>\n2
    \n<\/span><\/td>\n    		1
    \n<\/span><\/td>\n<\/tr>\n
    Puromycin<\/span><\/em><\/td>\n2.5 mg\/ml<\/span><\/td>\n2
    \n<\/span><\/td>\n    		1
    \n<\/span><\/td>\n<\/tr>\n
    Hygromycin<\/span><\/em><\/td>\n5 mg\/ml<\/span><\/td>\n2.5
    \n<\/span><\/td>\n    		1
    \n<\/span><\/td>\n<\/tr>\n
    Blasticidin<\/span><\/em><\/td>\n10 mg\/ml<\/span><\/td>\n10<\/span><\/td>\n5
    \n<\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

     <\/p>\n

    Protocol 2: nucleofection<\/strong><\/em><\/p>\n

    We use this protocol to integrate cassetes at RNA polymerase II transcribed loci; these typically represent much less efficient integration targets, and this approach is generally used for transfecting disruption or native tagging constructs<\/p>\n

      \n
    1. As per Protocol 1<\/em><\/strong><\/li>\n
    2. \u00a0\u00a0 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 -“-<\/li>\n
    3. \u00a0 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 -“-<\/li>\n
    4. Pour off the supernatant, remove as much of the residual media as possible and resuspend in 82 \u00b5l human T cell nucleofector solution, 18\u00a0\u00b5l supplement 1 (Lonza<\/a>; both at room temperatue) plus 10 \u00b5l DNA solution<\/li>\n
    5. Transfer cell-nucleofector solution-DNA mixture to a 2 mm gap cuvette and apply nucleofection programme X-001<\/li>\n
    6. As per Protocol 1<\/em><\/strong><\/li>\n
    7. \u00a0\u00a0 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 -“-
      \n<\/em><\/strong><\/li>\n<\/ol>\n

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

      We use two protocols to transfect linearised DNA constructs into bloodstream form T. brucei, depending…<\/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-873","page","type-page","status-publish","hentry","odd"],"yoast_head":"\nElectroporation - 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\/electroporation\/\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Electroporation - Alsford Lab\" \/>\n<meta property=\"og:description\" content=\"We use two protocols to transfect linearised DNA constructs into bloodstream form T. brucei, depending...\" \/>\n<meta property=\"og:url\" content=\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/electroporation\/\" \/>\n<meta property=\"og:site_name\" content=\"Alsford Lab\" \/>\n<meta property=\"article:modified_time\" content=\"2014-11-26T10:30:03+00:00\" \/>\n<meta name=\"twitter:label1\" content=\"Estimated reading time\" \/>\n\t<meta name=\"twitter:data1\" content=\"2 minutes\" \/>\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\/electroporation\/\",\"url\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/electroporation\/\",\"name\":\"Electroporation - 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