IGEM:Harvard/2010/General Protocols/Agrobacterium Electroporation

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*Press the "Settings" buttom to illuminate the LED next to "Manual". The display LED now shows voltage (in kV). Pressing the "Raise" and "Lower" buttons to the left of the display LED allows selection of the desired voltage. Agrobacterium require 2.5kV
*Press the "Settings" buttom to illuminate the LED next to "Manual". The display LED now shows voltage (in kV). Pressing the "Raise" and "Lower" buttons to the left of the display LED allows selection of the desired voltage. Agrobacterium require 2.5kV
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Electroporating
+
Electroporating:
*Electrocompetent bacterial cells (in -80 freezer), cuvettes, SOC media, and DNA solutions must all be kept on ice before mixing. The following steps should be carried out in under 1' and that you should be wearing glasses and gloves.
*Electrocompetent bacterial cells (in -80 freezer), cuvettes, SOC media, and DNA solutions must all be kept on ice before mixing. The following steps should be carried out in under 1' and that you should be wearing glasses and gloves.
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*Incubate at 28C and colonies should appear in 2-3 days
*Incubate at 28C and colonies should appear in 2-3 days
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Re-using cuvettes
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Re-using cuvettes:
*If you want to do this, fill a used cuvette with 0.1M H2SO4 and let stand 15min. Rinse 6x with dH20, then 2x with 96% EtOH. Store them well-covered in 70% EtOH.
*If you want to do this, fill a used cuvette with 0.1M H2SO4 and let stand 15min. Rinse 6x with dH20, then 2x with 96% EtOH. Store them well-covered in 70% EtOH.

Revision as of 14:14, 1 July 2010

Safety precautions:

  • Never change any other settings than stated while unit is charging (this may damage both the electroporator and powerssupply)
  • Keep unit away from water in a dry area and away from flammable materials
  • Never short circuit terminals
  • Whilst delivering pulse, keep hands away from chamber and cuvette. The result may otherwise be shocking

DNA preparations:

  • DNA for electroporation must be free of salt, RNA, or protein. To accomplish this, use Millipore Nitrocellulose Membrane Filters (Cat. No. VSWPO2500) and follow manufacturer's instructions

Preparing the electroporator (we used the MicroPulser from BioRad):

  • Use 2mm cuvettes from either Invitrogen
  • Connect black power cord
  • Pull down fold-down foot on underside of MicroPulser. Insert this foot into track on base of shocking chamber. Insert shocking chamber slide into shocking chamber.
  • Connect leads from shocking chamber to the output jacks on the front panel of MicroPulser
  • Turn on apparatus. The LED display should read "Ec1" and LED next to Bacteria Settings should be illuminated
  • Press the "Settings" buttom to illuminate the LED next to "Manual". The display LED now shows voltage (in kV). Pressing the "Raise" and "Lower" buttons to the left of the display LED allows selection of the desired voltage. Agrobacterium require 2.5kV

Electroporating:

  • Electrocompetent bacterial cells (in -80 freezer), cuvettes, SOC media, and DNA solutions must all be kept on ice before mixing. The following steps should be carried out in under 1' and that you should be wearing glasses and gloves.
  • Mix 1-2ul DNA (600ng) with 40ul cells
  • Transfer the DNA/cell mixture to a cuvette on ice avoiding air bubbles by gently shaking the cuvette
  • Dry outside of the cuvette with tissue paper and insert cuvette into the slide of the shocking chamber. Push the slide into the chamber until the cuvette makes firm contact with the chamber electrodes
  • To charge the capacitor and deliver a pulse, press the yellow "Pulse" button; the display LED will show "PLS" until a tone sounds indicating that the pulse has been given. The display LED will then show the program, the time constant, or the actual volts delivered, depending on the LED selected
  • Withdraw the slide from the chamber, remove cuvette, and process sample
  • With DNA/agro mix still in cuvette, add 500ml cold SOC (no antibiotics) and mix solution by gently pipetting up and down
  • Transfer cells to an eppi and incubate at 30 degrees for 2-4h
  • To turn the unit off, press the power switch on the right rear panel
  • Plate 200mL on LB+antibiotics
  • Incubate at 28C and colonies should appear in 2-3 days

Re-using cuvettes:

  • If you want to do this, fill a used cuvette with 0.1M H2SO4 and let stand 15min. Rinse 6x with dH20, then 2x with 96% EtOH. Store them well-covered in 70% EtOH.
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