NanoBio: Bacterial Transformation using Electroporation

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(Simultaneous Transformation using Electroporation)
Current revision (18:47, 12 June 2009) (view source)
(Simultaneous Transformation using Electroporation)
 
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==Simultaneous Transformation using Electroporation==
==Simultaneous Transformation using Electroporation==
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''If there are electrocompetent cells available to you in the -80°C, thaw the cells on ice and begin. If you need to make a fresh batch of electrocompetent cells, use either the small-scale or large-scale prep for electrocompetent cells.''
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''If there are electrocompetent cells available to you in the -80°C, thaw the cells on ice and begin. If you need to make a fresh batch of electrocompetent cells, use either the small-scale or large-scale *[[NanoBio: Prep for Electrocompetent cells | Prep for Electrocompetent cells]].''
## Chill cuvettes for at least 5 minutes on ice.
## Chill cuvettes for at least 5 minutes on ice.

Current revision

Simultaneous Transformation using Electroporation

If there are electrocompetent cells available to you in the -80°C, thaw the cells on ice and begin. If you need to make a fresh batch of electrocompetent cells, use either the small-scale or large-scale * Prep for Electrocompetent cells.

    1. Chill cuvettes for at least 5 minutes on ice.
    2. Set electroporator to 2.5kV. (Our electroporator is set to 600Ω)
    3. Add 5pg – 0.5ug DNA to cells. Mix by pipetting. Pippet into a sterile e.p. cuvette.
      • Note: This DNA should be salt-free, so when purifying with kit, use water.
      • Note: Include enough samples for +/- PCR.
      • Note: The DNA should not sit in the cells for more than 1 minute.
    4. Make sure to dry the cuvette to prevent arcing!
    5. Place the DRY cuvette into the sample chamber. Apply the pulse by pushing the pulse button twice. Remove the cuvette.
      • Note: If you hear a popping sound, this means that the sample has arced. This could either be (1) too much salt in your sample or (2) a wet cuvette.
    6. Immediately add 1mL of room-temp SOC or LB and transfer to a culture tube
    7. Incubate 1 hr with shaking at 37°C.
    8. Plate out cells on LB+antibiotic. Grow o/n at 37°C.


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