User:Kalkao/notebook/Preparing Competent Cells for Phage Transfection
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This protocol was developed from the supplementary information of the "Refactoring bacteriophage T7" paper.
- Obtain IJ1126 overnights and warmed LB from the 37*C incubation room.
- 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.
- Place a bottle of 50mM CaCl2 on ice.
- 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.
- Obtain two plastic 40mL centrifuge tubes pipette all of the IJ1126 culture out of the flask and into one of the plastic centrifuge tubes.
- When the centrifuge is finished, remove the tubes.
- 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.
- Pour the supernatant of the culture-containing tube into the sink
- Allow this new suspension to incubate in the ice bucket in the 4*C fridge for 30 minutes.
- 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.
- 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.