NanoBio: Prep for Electrocompetent cells: Difference between revisions

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(New page: =Small-scale prep= =Large-scale prep= *'''~~~~''': Return to Protocols.)
 
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''There are a couple of tricks to making cells electrocompetent.''
''#Remove as much salt from the cell suspensions as possible to prevent arcing. ''
''#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. ''
=Small-scale prep=
=Small-scale prep=
# 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.
Return to [[NanoBio:Protocols|Protocols]].


=Large-scale prep=
=Large-scale prep=

Revision as of 15:25, 12 June 2009

There are a couple of tricks to making cells electrocompetent. #Remove as much salt from the cell suspensions as possible to prevent arcing. #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.

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.
    • Note: Include enough samples for +/- L-arabinose induction
  3. When OD600 = 0.1, add 20uL of L-arabinose stock to induce pKD46 λ-red expression
  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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