User:Kalkao/notebook/Preparing Competent Cells for Phage Transfection

From OpenWetWare

Jump to: navigation, search

This protocol was developed from the supplementary information of the "Refactoring bacteriophage T7" paper.

  1. Obtain IJ1126 overnights and warmed LB from the 37*C incubation room.
  2. Use two armed flasks to grow cells to a density of approximately 5e8 cells/mL.
    • Pipette 20mL of warm LB into each flask.
    • Add 20uL of overnight culture to one flask for a 1:1000 dilution.
    • Incubate this flask in the 30*C shaking water bath.
    • Check the OD periodically using the Using the Klett Photoelectric Colorimeter protocol.
    • When the culture reaches approximately 60 klett, remove the flask and place it on ice.
      • The culture will follow an exponential growth pattern, so the OD will begin to increase very quickly as it nears 60 klett.
  3. Place a bottle of 50mM CaCl2 on ice.
  4. Pellet the cells:
    • Obtain two plastic 40mL centrifuge tubes pipette all of the IJ1126 culture out of the flask and into one of the plastic centrifuge tubes.
      • Keep the culture on ice whenever possible.
    • Use a beaker to hold the tube with culture on the weight plate and tare the beaker and centrifuge tube together.
    • Remove the tube containing culture and replace it with the other tube. Leave this tube open (and include the cap in the beaker).
    • Pour water into the tube until the mass balance reads zero.
    • Use the SA600 rotor to centrifuge the tubes at 5,000 RPM for 5 minutes.
      • Make sure to tighten both knobs when putting the cover on the rotor.
      • Wait until the centrifuge is up to speed to set the timer to 5min.
  5. When the centrifuge is finished, remove the tubes.
  6. Resuspend the pellet in cold 50mM CaCl2.
    • Pour the supernatant of the culture-containing tube into the sink
      • Touch the lip of the tube to a paper towel to take away remaining liquid.
    • Resuspend the pellet in 10mL of 50mM CaCl2 and place the tube on ice.
      • The amount of 50mM CaCl2 should correspond to half of the volume of the starting culture.\
    • Be sure to label this tube with a sticker because both liquids will now look the same.
  7. Allow this new suspension to incubate in the ice bucket in the 4*C fridge for 30 minutes.
  8. After the 30 minute incubation, pellet the cells again at 5,000 RPM for 5 minutes.
    • Be sure to weigh out a new balance centrifuge tube.
  9. Resuspend the new pellet in cold 50mM CaCl2.
    • Pour the supernatant into the sink.
    • Touch the lip of the tube to a paper towel to soak up remaining CaCl2.
    • Resuspend the cells in 2mL of CaCl2.
      • The new amount of CaCl2 should correspondg to one-tenth of the original culture volume.
    • Put this suspension back on ice and put the ice bucket in the 4*C fridge.
      • Incubate this suspension overnight.
      • Tomorrow, the cells should be ready for transfection.
Personal tools