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

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