Dicty Transformation

Preparation

  • You will need enough cells...
    • A fully confluent 10cm plate will give enough for 2-3 transformations
    • A fully confluent 15cm plate will give 5-10
    • For maintenace of cell line, the cells should be passed just prior to the transformation (transfer 100ml of cells to fresh plate with 10mls HL5+P/S(6ml/ml)+Thy(0.1mg/ml))
  • You neede to have enough DNA at a reasonable concentration...
    • Each transformation requires 5mg of DNA
    • The 5ug also must be in a volume between 5-15ml, the concentrations can be adjusted by dilution or ethanol precipitation

Procedure

  1. Obtain fresh (sterile) electroporation (EP) cuvettes (0.4cm-width) and place on ice.
  2. Spot 5mg of DNA to be transformed on the side of one of the sterile cuvettes and leave on ice. Note: A volume between 5-15ml should give the 5mg. If not adjust by dilution or ethanol precipitation.
  3. Obtain a plate of confluent cells, subculture 100ml, and then resuspend all cells in current media using a pipette to rinse cells off plate. Transfer all resuspended cells in a 15ml orange cap tube. Note: The room temperature media is used for resuspension because cold solutions make resuspension difficult.
  4. Centrifuge at 2000RPM for 2minutes, and then aspirate off entire supernatant with sterile pasteur pipette.
  5. Gently resuspend cells in appropriate amount of EP buffer (10mM KH2PO4 50mM Sucrose pH 6.5). - 2-3 transformations per 10cm plate, 350ml of EP buffer per transformation
  6. Wash spot of DNA off side of EP cuvette with 350ml of cells in EP buffer.
  7. Zap cells using electroporator at 1.3kV and 3mF with resistor unhooked, make sure to record time constants (should be between 1.8-2.2)
  8. Plate 50 and 200ml of zapped cells onto fresh plates with 10mls HL5+Thy+P/S. Swirl to distribute cells
  9. 12-24 hours later add desired selective agent (i.e. G418 at 5mg/ml).