NanoBio: Bacterial Transformation using Electroporation: Difference between revisions

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(New page: ==Simultaneous Transformation using Electroporation== Note: this protocol may be used for any cell line, but may have to be optimized. ''There are a couple of tricks to making cells elec...)
 
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==Simultaneous Transformation using Electroporation==
==Simultaneous Transformation using Electroporation==


Note: this protocol may be used for any cell line, but may have to be optimized.
''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.''


''There are a couple of tricks to making cells electrocompetent. (1) Remove as much salt from the cell suspensions as possible to prevent arcing. (2) The cells must remain cold (either 4°C cold room or on ice).  Once in DI water, the cells become very sensitive to temperature changes and the transformation efficiency drops dramatically if cells are allowed to warm up above 4°C at any step. It is also better to make your cells "fresh" so we only do small volumes of cells.''
# Make cells electrocompetent.
## Pre-chill all tubes, solutions, and cuvettes!
## Pick some colonies from a fresh plate (or back dilute with 20uL from o/n culture) and grow at 30degC in 2mL LB+Amp.
##* ''Note: Include enough samples for +/- L-arabinose induction''
## When OD600 = 0.1, add 20uL of L-arabinose stock to induce pKD46 λ-red expression
## Continue growing at 30degC until OD600 = 0.4 - 0.6
## Aliquot 1mL from each sample into 2x 1.5mL centrifuge tubes
## Chill cells in ice-water bath 10-15min
## Centrifuge 10m at 4000rcf at 4°C
##* ''Note: the centrifuge next to the bioflo cabinet has temp control''
## Pipette/discard supernatant and resuspend cells (GENTLY) in 1mL ice-cold dH2O
## Centrifuge 10m at 4000rcf at 4°C
## Resuspend cells (GENTLY) in 0.5mL ice-cold dH2O
##* ''Note: after washing in DI water, the cells become more and more difficult to spin down. Using 10% glycerol can help spin down cells''
## Centrifuge 10m at 4000rcf at 4°C
## Resuspend pellet (GENTLY) in 50uL ice-cold water.
# Finally, electroporate.
## Chill cuvettes for at least 5 minutes on ice.
## Chill cuvettes for at least 5 minutes on ice.
## Set electroporator to 2.5kV. (Our electroporator is set to 600Ω)
## Set electroporator to 2.5kV. (Our electroporator is set to 600Ω)

Revision as of 15:21, 12 June 2009

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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