Competent Cell Preparation

Supplies:

• DH5(alpha)
• LB broth--about 50mL per culture (each culture makes about 25-30 * 100 uL aliquots); about 5 mL for the initial overnight culture (inoculates multiple cultures)
• 0.1M CaCl2 (filter sterilized and chilled)--about 40-50 mL per culture
• Ice water bath
• 60% glycerol--about 1 mL per culture
• Spectrophotometer
• 100-250 mL flasks, centrifuge tubes (50 mL Falcon or equivalent), microcentrifuge tubes Note: When working with cultures (that you will use later), always work aseptically. That is, work near a flame and be sure to turn off the flame at the end and follow aseptic procedures.

Steps:

1. Inoculate 5mL overnight culture (see overnight culture protocol) and grow at 30°C. You can use AMBL lab rotating incubator). Approximately 30°C (can be a little higher or lower) seems to be fine. Otherwise, shaking speed ~200 rpm. Note:A possible source of DH5(alpha) are frozen aliquots of DH5(alpha) competent cells. The whole aliquot.


2. Dilute 0.5mL overnight culture into 50mL LB and incubate at 30°C, shaking vigorously. Label with name, date, strain, flask # (when making more than 1 culture), etc. Finlay Lab common incubator can be used for this (the one that says to not change the settings). Shaking speed ~200 rpm. Note: Several 50 mL LB cultures can be inoculated and used at the same time. A 125 mL or 250 mL Erlenmeyer flask works well as a culturing container. Make sure to not rip or contaminate the aluminum foil cover. When done using the flasks, rinse with 10% bleach, then with water and put into dirty basket (ask Tony Lam for help if needed).


3. Harvest at Abs600nm = 0.401. Higher than this seems to work well anyway. Pipet the cultures into 50 mL Falcon tubes (don’t overfill).
   
   • Using a spectrophotometer: Take 1 mL from the culture and 1 mL from a LB stock and put into cuvettes. Turn on the spectrophotometer (on Antonio’s bench). Wipe the cuvette with Kimwipe (to clean the part where light passes through). Press the OD600 button. Put the 1 mL LB cuvette into the slot and press the Blank button. Replace the 1 mL LB cuvette with 1 mL culture cuvette and press sample. Rinse the cuvettes twice with 70% ethanol and twice with sdH20. Turn off spectrophotometer.
   Note: No need to work aseptically when using the spectrophotometer. However, as always, transferring cultures that you want to use later on means you should work aseptically.
4. Centrifuge at 1600g for 7 minutes (4°C). Kronstad lab common centrifuge can be used for this.
   • Kronstad lab centrifuge: Set the rotor type (e.g. 5.3), RCM (e.g. 1600 g), temperature =4°C, and time. Then press start (if machine is off, turn on). In any case, follow the instructions on the centrifuge. Make sure the samples are balanced. You may leave the temperature at 4°C (the machine will keep it nice for you). If someone else is going to use it, return the temperature to about 21°C. Turn off the centrifuge when you won’t use it again. Sign your name on the notepad. Emergency contact details are near the centrifuge.
5. Pour out the supernatant aseptically (also flame mouth and cap before and after). Wash gently in ~20mL COLD filter sterilized 0.1M CaCl2 (i.e. put the tube on ice or put in cooler). Swirl the tube using your wrist until the pellet disappears.
   • Filter sterilization: Use a syringe to suck up some CaCl2 (from a beaker probably). Attach a 0.4 micron filter unit to the tip. Press the plunger. Whether any part above the filter is sterile is unimportant; anything that passes through the filter is sterile. Make sure the filtrate enters a sterile container (e.g. Falcon tube). You can re-use the filter: remove the filter, use the syringe to suck up more CaCl2 and repeat. The original packaging material can hold the filter while you do this. Make sure the first few drops of filtrate does not go into the container with your previously sterilized CaCl2. You can drop it back into the unsterilized CaCl2. Label finished product.


6. Spin down gently: 1100g, 5 minutes, 4°C. See 4 about using the centrifuge.


7. Pour out the supernatant aseptically (flame mouth and cap before and after too).


8. Resuspend in 12.5mL cold 0.1M CaCl2. Wrist.


9. Keep on ice 40 minutes.


10. Spin down: 1100g, 5 minutes, 4°C. See 4 about using the centrifuge.


11. Pour out the supernatant aseptical (flame mouth and cap before and after too). Resuspend in 2-2.5mL cold 0.1M CaCl2. Wrist.


12. Store over night at 4°C.


13. Add glycerol to 15% and divide into 100uL aliquots, store at -80°C in Lagally Lab freezer; label with name, date, strain, flask where it came from, etc. Adding about 1 mL 60% glycerol to 2.5 mL of the final resuspended cells yields a ~15% glycerol solution.


14. Throw away used tubes, pipets, filters, syringes, etc.