NanoBio: 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). 
 * 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 37°C in 2mL LB+antibiotics.
 * 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
 * 8) * Note: the centrifuge next to the bioflo cabinet has temp control
 * 9) Pipette/discard supernatant and resuspend cells (GENTLY) in 1mL ice-cold dH2O
 * 10) Centrifuge 10m at 4000rcf at 4°C
 * 11) Resuspend cells (GENTLY) in 0.5mL ice-cold dH2O
 * 12) * Note: after washing in DI water, the cells become more and more difficult to spin down. Using 10% glycerol can help spin down cells
 * 13) Centrifuge 10m at 4000rcf at 4°C
 * 14) Resuspend pellet (GENTLY) in 50uL ice-cold water.

Return to Protocols.

=Large-Scale prep=

''Rinse all flasks with H2O prior to autoclaving in order to remove residual detergents/salts that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better. ''


 * 1) Pre-chill all tubes and solutions! This should include the box and tubes for the cells to be stored in (chill in -80°C)
 * 2) Grow an o/n culture of the strain with antibiotics, if appropriate, in LB at 37°C
 * 3) Inoculate 500mL fresh LB with the 5mL o/n culture. Put it back on the shaker at 37°C.
 * 4) Grow until the OD600 is between 0.4 - 0.6. (~3 hours)
 * 5) Aliquot 35mL from each sample into 16x 40mL centrifuge tubes.
 * 6) Chill cells in ice-water bath 15 - 60 min. Increased chill time may increase competency.
 * 7) *This will subsequently be broken into two batches of 8 tubes. Chill the first batch for 40 minutes. The second batch for 60 minutes. This should give a good rhythm of washing and spinning.
 * 8) Spin #1: Centrifuge 10m at 4000rcf at 4°C
 * 9) Discard supernatant and resuspend cells in 35mL ice-cold dH2O.
 * 10) *Resuspend gently in 5-10 mL and then add remaining volume.
 * 11) Spin #2: Centrifuge 15m at 4000rcf at 4°C
 * 12) Discard supernatant and resuspend cells (GENTLY) in 25mL ice-cold dH2O.
 * 13) *Resuspend gently in 5-10 mL and then add remaining volume.
 * 14) Chill on ice for 30 minutes.
 * 15) Spin #3: Centrifuge 15m at 4000rcf at 4°C
 * 16) Discard supernatant and resuspend cells in 3.5mL ice-cold 10% glycerol.
 * 17) *This will not be a good pellet. Be careful when discarding supernatant. (Use pipette. Do not pour off.)
 * 18) Chill on ice for 30 minutes.
 * 19) Spin #4: Centrifuge 15m at 4000rcf at 4°C
 * 20) Remove the supernatant and add 100 μl of 10% glycerol.
 * 21) Add 10% glycerol to a total volume of ~160μL if necessary. (probably won't be!)
 * 22) Aliquot 50μL of cell suspensions in pre-chilled tubes.
 * 23) Shock freeze the tubes in a mixture of dry ice and ethanol.
 * 24) Store at -80°C. These will last between 3-6 months.
 * 25) When ready to use, thaw on ice and use immediately.

Return to Protocols.
 * Heather M. Jensen 18:23, 12 June 2009 (EDT):