NanoBio: Prep for Electrocompetent cells

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Large-Scale prep)
Current revision (13:21, 15 June 2009) (view source)
(Large-Scale prep)
 
Line 26: Line 26:
=Large-Scale prep=
=Large-Scale prep=
-
''Rinse all flasks with H<sub>2</sub>OH2O 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. ''
+
''Rinse all flasks with H<sub>2</sub>O 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. ''
# Pre-chill all tubes and solutions! This should include the box and tubes for the cells to be stored in (chill in -80°C)
# Pre-chill all tubes and solutions! This should include the box and tubes for the cells to be stored in (chill in -80°C)

Current revision

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
    • 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

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.
    • 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.
  7. Spin #1: Centrifuge 10m at 4000rcf at 4°C
  8. Discard supernatant and resuspend cells in 35mL ice-cold dH2O.
    • Resuspend gently in 5-10 mL and then add remaining volume.
  9. Spin #2: Centrifuge 15m at 4000rcf at 4°C
  10. Discard supernatant and resuspend cells (GENTLY) in 25mL ice-cold dH2O.
    • Resuspend gently in 5-10 mL and then add remaining volume.
  11. Chill on ice for 30 minutes.
  12. Spin #3: Centrifuge 15m at 4000rcf at 4°C
  13. Discard supernatant and resuspend cells in 3.5mL ice-cold 10% glycerol.
    • This will not be a good pellet. Be careful when discarding supernatant. (Use pipette. Do not pour off.)
  14. Chill on ice for 30 minutes.
  15. Spin #4: Centrifuge 15m at 4000rcf at 4°C
  16. Remove the supernatant and add 100 μl of 10% glycerol.
  17. Add 10% glycerol to a total volume of ~160μL if necessary. (probably won't be!)
  18. Aliquot 50μL of cell suspensions in pre-chilled tubes.
  19. Shock freeze the tubes in a mixture of dry ice and ethanol.
  20. Store at -80°C. These will last between 3-6 months.
  21. When ready to use, thaw on ice and use immediately.


Return to Protocols.

Personal tools