Tranformatio of ES cells using Electroporation

Overview

This protocol describes how to transform ES cells using electroporation. Electroporation will probably kill about half the cells and about 2 to 20% of the remaining cells will be successfully transfected.

Protocol

• Start with a 10 cm plate of almost confluent ES cells.
• If trying to get stable integration, linearize the plasmid before electroporation.
• Three hours before electroporation change media in plates
• One hour before electroporation warm media and gather supplies
• Prepare four plates for each transfection by coating with gelatin and adding 5 ml media
• Note: If maintaining as stem cells, these should have irradiated MEFs also
• Trypsinze ES cells with 4 ml trypsin/EDTA per 10 cm plate for 5 minutes at 37ºC
• Add 10 ml ES media, transfer to 15 ml conical and spin 5 min 1000 rpm
• Remove supernatant and resuspend in 10 ml PBS (no Ca++/Mg++)
• Count cells
• Spin 5 min 1000 rpm. Remove supernatant and resuspend at 1e7 cells/ml in PBS
• Aliquot 0.8 ml cell solution into a 0.4 mm cuvette (control) (8e6 cells)
• Aliquot 0.8 ml cell solution with 20 - 25 µg of linearized DNA at 1 µg/ml in 0.4 mm cuvette
• Note: Make sure there are no bubbles in the cuvette
• Incubate RT 5 min
• Electroporate with the following settings:
  • 250 V 400 µf
  • Resistance at infinity
  • Cuvette 4
  • Note down the time constant (should be 7 to 8)
• Incubate RT 5 min
• Note: There should be bubbles on top from shock and exploded cells floating on top
• Transfer cuvette contents to 50 ml conical containing 20 ml prewarmed media
• Aliquot 5 µl to each of the 4 gelatin coated plates already containing 5 ml media
• Incubate 24 hours before adding selection agent
• Change media every day
• Harvest at 48 to 72 hours