Content Loading...
Content Loading...
Your Cart

0 Items

Favorites

0 Items

Last Item Added: 0 Items
Content Loading...
» Close

Electroporation of ES Cells


Protocol for Serum-containing Medium

Materials and Reagents
  • Electroporator and 0.4 cm cuvette
  • EmbryoMax ES Cell Qualified Electroporation Buffer - Millipore cat. no. ES-003-D
  • ES Cell Medium • Ice • Incubator, 37°C/5% CO2
  • 25–40 µg Linearized construct DNA, ethanol precipitated and dried
  • PMEF Feeder cell coated plates
  • 0.05% Trypsin-0.53 mM EDTA - Millipore cat. no. SM-2002-C
Protocol
  1. The evening before the electroporation is to be performed, prepare 4 plates with PMEF cells.
  2. The morning that the electroporation is to be performed, feed the ES cells with fresh ES Cell Medium.
  3. Later that afternoon, harvest the ES cells and determine the cell count. 1 x 107 ES cells is the minimum number of ES cells required for electroporation. If there is excess, freeze the cells down.
  4. Centrifuge the cells required for electroporation at 300 xg for 10 minutes, then aspirate the medium.
  5. Resuspend the ES cell pellet in 600 µL of Electroporation Buffer.
  6. 25–40 µg of knockout construct DNA (purified) should already be linearized, ethanol precipitated and dried as a pellet. In a sterile hood, dissolve the DNA pellet in 30 µL of Electroporation Buffer, and then add the solution to the ES cells. Mix well and leave for 5 minutes at room temperature.
  7. Place the ES cells in a 0.4 cm electroporation cuvette. Electroporate the suspension at 500 µFD, 0.24 kV. The time constant produced should be between 6.9 and 7.9 milliseconds (optimal 7.2). Following electroporation, place the cuvette on ice for 10 minutes.
  8. Transfer the electroporated ES cells to 40 mL of ES Cell Medium and mix gently using a Pasteur pipette.
  9. Plate the ES cell suspension (10 mL per feeder plate, total of 4 plates). Ensure that the PMEF Feeder Cell Medium is removed prior to the addition of cells.
  10. Incubate for approximately 36 hours at 37°C and 5% CO2 prior to antibiotic selection.

Protocol for Serum-free Medium

Materials and Reagent
  • EmbryoMax ES Cell Qualified Electroporation Buffer — Millipore cat. no. ES-003-D
  • ESGRO Complete Clonal Grade Medium — Millipore cat. no. SF001-500
  • Accutase Solution for ESGRO Complete system — Millipore cat. no. SF006
  • 25–40 µg Linearized construct DNA, ethanol precipitated and dried
  • Incubator, 37°C/5% CO2
  • Electroporator and 0.4 cm cuvette
Protocol
  1. The morning that the electroporation is to be performed, feed the ES cells with fresh ESGRO Complete Clonal Grade Medium.
  2. Later that afternoon, prepare 4 plates with 0.1% ESGRO Complete Gelatin solution. Harvest the ES cells and determine the cell count. The minimum number of ES cells required for electroporation is 1 x 107 cells. If there are excess cells, freeze them down.
  3. Centrifuge the cells required for electroporation at 300 xg for 3 minutes, then aspirate the medium.
  4. Resuspend the ES cell pellet in 600 µL of Electroporation Buffer.
  5. 25–40 µg of knockout construct DNA (purified) should already be linearized, ethanol precipitated and dried as a pellet. In a sterile hood, dissolve the DNA pellet in 30 µL of Electroporation Buffer, and then add the solution to the ES cells. Mix well and leave for 5 minutes at room temperature.
  6. Place the ES cells in a 0.4 cm electroporation cuvette. Electroporate the suspension at 3 µFD, 0.8 kV (BioRad Gene Pulser — if other instrument is used, then optimal times and voltage must be determined). The time constant produced should be between 6.9 and 7.9 milliseconds (optimal 7.2). Following electroporation, leave the cuvette at room temperature for 10 minutes.
  7. Transfer the electroporated ES cells to 40 mL of Clonal Grade Medium and mix gently using a Pasteur pipette.
  8. Plate the ES cell suspension (10 mL per plate, total of 4 plates). Ensure that the 0.1% Gelatin solution is removed prior to the addition of cells.
  9. Incubate for approximately 36 hours at 37°C and 5% CO2 prior to antibiotic selection.

back to top

Find Primary Antibodies

Enter a keyword and/or search criteria below. Or you may also use selections below to browse specific antibodies for your needs.

Product News & Releases

» View All

Featured Products



Pathways