{"id":569,"date":"2017-10-18T13:19:41","date_gmt":"2017-10-18T20:19:41","guid":{"rendered":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/?page_id=569"},"modified":"2023-04-27T12:34:37","modified_gmt":"2023-04-27T19:34:37","slug":"protocol-9-electroporation-of-20-zr","status":"publish","type":"page","link":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/protocol-9-electroporation-of-20-zr\/","title":{"rendered":"Protocol 9: Electroporation of 20 ZR"},"content":{"rendered":"\n

Electroporation of 20ZR<\/sup><\/u><\/strong><\/p>\n\n\n\n

Day 1: <\/p>\n\n\n\n

    \n
  1. Inoculate 2-3 bottles (with 25ml P 0.75% media) with 20ZR<\/sup><\/li>\n\n\n\n
  2. Add methane (~40-45cc) to the bottles and incubate at 30o<\/sup>C until the OD is between 0.3 and 0.6 at 600 nm<\/li>\n<\/ol>\n\n\n\n

    Day 2: <\/p>\n\n\n\n

      \n
    1. Once the OD is between 0.3 and 0.6 at 600 nm, pool the three 20ZR<\/sup> cultures and divide them into two 50.0 ml centrifuge tubes <\/li>\n\n\n\n
    2. Centrifuge the 20ZR<\/sup> culture at 4700 rpm for 15 min at 4o<\/sup>C<\/li>\n\n\n\n
    3. Remove and discard the supernatant without disturbing the pellet (NOTE: I decant the supernatant) <\/li>\n\n\n\n
    4. Re-suspend the pellets in a total of 10 ml room temperature DI H2<\/sub>O (milliQ water) and transfer to 10.0 ml centrifuge tube. <\/li>\n\n\n\n
    5. Centrifuge for 5 min at 4700 rpm at 4o<\/sup>C.<\/li>\n\n\n\n
    6. Discard the supernatant without disturbing the pellet. Add 5 ml of DI H2<\/sub>O to the pellet but DO NOT suspend the pellet. <\/li>\n\n\n\n
    7. Centrifuge for 5 min at 4700 rpm at 4o<\/sup>C.<\/li>\n\n\n\n
    8. Discard the supernatant without disturbing the pellet. Wash the pellet with 5 ml DI H2<\/sub>O. DO NOT centrifuge \uf0e0discard the wash. <\/li>\n\n\n\n
    9. Re-suspend the pellet in 100-150ml DI water (you will need 50ml\/transformation) and place on ice. **From this point on, everything should be kept on ice until the electroporation is completed <\/strong><\/li>\n\n\n\n
    10. Transfer 50ml of cell suspension into an eppendorf tube <\/li>\n\n\n\n
    11. Add DNA (less than 6ml of DNA, ~300-500 ng DNA ) to appropriate tubes and mix gently <\/li>\n\n\n\n
    12. Transfer to an ice-cold 0.1cm gap electroporation cuvette <\/li>\n\n\n\n
    13. Electroporate with 1.5kV, 200 \u03a9, 25mF (time constant is usually 4.5-5ms) and immediately add 1ml room temperature P3% media. <\/li>\n\n\n\n
    14. Transfer the electroporated cells into 10ml P3% in 125ml bottles.<\/li>\n\n\n\n
    15. Add 40 cc methane and incubate the cultures at 30o<\/sup>C shaking incubator for overnight (~14-16 hrs). <\/li>\n<\/ol>\n\n\n\n

      Day 3: <\/p>\n\n\n\n

      \u00b7 Centrifuge cells for 15 min at room temperature at 4700 rpm <\/p>\n\n\n\n

      \u00b7 Re-suspend cells in 500ml-1ml P3% media  <\/p>\n\n\n\n

      \u00b7 Plate each suspension on 3-4 selective plates with appropriate antibiotics <\/p>\n","protected":false},"excerpt":{"rendered":"

      Electroporation of 20ZR Day 1:  Day 2:  Day 3:  \u00b7 Centrifuge cells for 15 min at room temperature at 4700 rpm  \u00b7 Re-suspend cells in 500ml-1ml P3% media   \u00b7 Plate each suspension on 3-4 selective plates with appropriate antibiotics <\/p>\n","protected":false},"author":3,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"off","_et_pb_old_content":"\n\n\n

      <\/p>\n","_et_gb_content_width":"","footnotes":""},"class_list":["post-569","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/pages\/569","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/comments?post=569"}],"version-history":[{"count":5,"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/pages\/569\/revisions"}],"predecessor-version":[{"id":1411,"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/pages\/569\/revisions\/1411"}],"wp:attachment":[{"href":"https:\/\/sci.sdsu.edu\/kalyuzhlab\/wp-json\/wp\/v2\/media?parent=569"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}