Journal article Open Access

Electroporation of ES cells

sprotocols


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{
  "publisher": "Zenodo", 
  "DOI": "10.5281/zenodo.13636", 
  "author": [
    {
      "given": "", 
      "family": "sprotocols"
    }
  ], 
  "issued": {
    "date-parts": [
      [
        2014, 
        12, 
        30
      ]
    ]
  }, 
  "abstract": "Author: Nagy Lab\n\nCells are routinely passaged two days prior to electroporating. Usually one 10 cm plate at approximately 80% confluency will provide enough cells for 1-2 electroporations.\n\n### Procedure\n\n1. Change medium on ES cells 3-4 hours prior to electroporation\n- Gelatinize 10 cm plates, then add 10 ml medium to each.\n- Place them in a 37 0C incubator until they are required.\n- Switch on the electroporation apparatus.\n- Harvest ES cells by trypsinization.\n- Resuspend the cell pellet in ice-cold PBS (1 ml for each 10 cm plate).\n- Determine the cell density (haemocytometer) and dilute with PBS to the required density for electroporation. We regularly electroporate at a relatively high cell density: 7x106 cells/ml (this number varies between different labs).\n- For each electroporation mix together 20-40 microgram (1 \u00b5g/\u00b5l) DNA (for an approximately 10 kb vector) and 0.8 ml of the ES cell suspension in an electroporation cuvette (BioRad, Cat. No. 165-2088).\n- Set up the electroporation conditions prior to placing the cuvette into the electroporation chamber. We routinely use 250 V, 500 microF for the BioRad GenePulser.\n-  Zap the cuvette, then place it on ice for 20 min to 1 hour.\n- Transfer the cells from the cuvette into the prewarmed medium containing dishes. (The contents of one cuvette are routinely seeded into two 10 cm dishes).\n\n**post electroporation**:\n\n12.Change medium daily.\n\n13.If drug selection is required start this on the second day after electroporation.\n\n14.Continue the selection until colonies become apparent, and grow to a size that is amenable to picking (usually takes 7-10 days).", 
  "title": "Electroporation of ES cells", 
  "type": "article-journal", 
  "id": "13636"
}
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