Journal article Open Access

Electroporation of ES cells

sprotocols


DataCite XML Export

<?xml version='1.0' encoding='utf-8'?>
<resource xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns="http://datacite.org/schema/kernel-4" xsi:schemaLocation="http://datacite.org/schema/kernel-4 http://schema.datacite.org/meta/kernel-4.1/metadata.xsd">
  <identifier identifierType="DOI">10.5281/zenodo.13636</identifier>
  <creators>
    <creator>
      <creatorName>sprotocols</creatorName>
      <affiliation>ScientificProtocols.org</affiliation>
    </creator>
  </creators>
  <titles>
    <title>Electroporation of ES cells</title>
  </titles>
  <publisher>Zenodo</publisher>
  <publicationYear>2014</publicationYear>
  <dates>
    <date dateType="Issued">2014-12-30</date>
  </dates>
  <resourceType resourceTypeGeneral="JournalArticle"/>
  <alternateIdentifiers>
    <alternateIdentifier alternateIdentifierType="url">https://zenodo.org/record/13636</alternateIdentifier>
  </alternateIdentifiers>
  <rightsList>
    <rights rightsURI="https://creativecommons.org/publicdomain/zero/1.0/legalcode">Creative Commons Zero v1.0 Universal</rights>
    <rights rightsURI="info:eu-repo/semantics/openAccess">Open Access</rights>
  </rightsList>
  <descriptions>
    <description descriptionType="Abstract">Author: Nagy Lab

Cells 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.

### Procedure

1. Change medium on ES cells 3-4 hours prior to electroporation
- Gelatinize 10 cm plates, then add 10 ml medium to each.
- Place them in a 37 0C incubator until they are required.
- Switch on the electroporation apparatus.
- Harvest ES cells by trypsinization.
- Resuspend the cell pellet in ice-cold PBS (1 ml for each 10 cm plate).
- 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).
- For each electroporation mix together 20-40 microgram (1 µg/µl) 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).
- 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.
-  Zap the cuvette, then place it on ice for 20 min to 1 hour.
- 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).

**post electroporation**:

12.Change medium daily.

13.If drug selection is required start this on the second day after electroporation.

14.Continue the selection until colonies become apparent, and grow to a size that is amenable to picking (usually takes 7-10 days).</description>
  </descriptions>
</resource>
24
6
views
downloads
All versions This version
Views 2424
Downloads 66
Data volume 9.8 kB9.8 kB
Unique views 2222
Unique downloads 66

Share

Cite as