NanoBio: Prep for Electrocompetent cells

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(Small-scale prep)
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# Pre-chill all tubes, solutions, and cuvettes!  
# 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.  
# Pick some colonies from a fresh plate (or back dilute with 20uL from o/n culture) and grow at 30degC in 2mL LB+Amp.  
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#* ''Note: Include enough samples for +/- L-arabinose induction''
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# If colonies need to be induced, add inducer at OD600 = 0.1
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# When OD600 = 0.1, add 20uL of L-arabinose stock to induce pKD46 λ-red expression
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# Continue growing at 30degC until OD600 = 0.4 - 0.6
# Continue growing at 30degC until OD600 = 0.4 - 0.6
# Aliquot 1mL from each sample into 2x 1.5mL centrifuge tubes
# Aliquot 1mL from each sample into 2x 1.5mL centrifuge tubes
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Return to [[NanoBio:Protocols|Protocols]].
Return to [[NanoBio:Protocols|Protocols]].
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=Large-Scale prep=
=Large-Scale prep=

Revision as of 18:29, 12 June 2009

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).
  3. 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.

Small-scale prep

  1. Pre-chill all tubes, solutions, and cuvettes!
  2. Pick some colonies from a fresh plate (or back dilute with 20uL from o/n culture) and grow at 30degC in 2mL LB+Amp.
  3. If colonies need to be induced, add inducer at OD600 = 0.1
  4. Continue growing at 30degC until OD600 = 0.4 - 0.6
  5. Aliquot 1mL from each sample into 2x 1.5mL centrifuge tubes
  6. Chill cells in ice-water bath 10-15min
  7. Centrifuge 10m at 4000rcf at 4°C
    • Note: the centrifuge next to the bioflo cabinet has temp control
  8. Pipette/discard supernatant and resuspend cells (GENTLY) in 1mL ice-cold dH2O
  9. Centrifuge 10m at 4000rcf at 4°C
  10. 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
  11. Centrifuge 10m at 4000rcf at 4°C
  12. Resuspend pellet (GENTLY) in 50uL ice-cold water.


Return to Protocols.

Large-Scale prep

Return to Protocols.

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