NanoBio: Prep for Electrocompetent cells: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
Line 1: | Line 1: | ||
''There are a couple of tricks to making cells electrocompetent.'' | ''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= | |||
# Pre-chill all tubes, solutions, and cuvettes! | # Pre-chill all tubes, solutions, and cuvettes! | ||
Line 26: | Line 26: | ||
=Large-Scale prep= | |||
*'''[[User:Heather M. Jensen|Heather M. Jensen]] 18:23, 12 June 2009 (EDT)''': | *'''[[User:Heather M. Jensen|Heather M. Jensen]] 18:23, 12 June 2009 (EDT)''': | ||
Return to [[NanoBio:Protocols|Protocols]]. | Return to [[NanoBio:Protocols|Protocols]]. |
Revision as of 15:27, 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
- 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 Protocols.
Large-Scale prep
- Heather M. Jensen 18:23, 12 June 2009 (EDT):
Return to Protocols.