Calcium chloride competent cells-PDF

The following protocol is for the preparation of chemically competent E. coli using calcium chloride. It is adapted from Current Protocols in Molecular Biology:

Seidman, Christine E. “Basic Protocol 1: Transformation Using Calcium Chloride.” UNIT 1.8 Introduction of Plasmid DNA into Cells. Current Protocols in Molecular Biology, 2005. http://www3.interscience.wiley.com/cgi-bin/mrwhome/104554809/HOME

Materials

• Overnight E. coli culture
• LB
• CaCl2 solution, ice cold
• Chilled 50 ml polypropylene tubes

CaCl2 solution:

• 60 mM CaCl2
• 15% glycerol
• 10 mM PIPES
  pH 7. Filter sterilize and store at room temp.

Procedure

1. Subculture an E.coli overnight culture 1:100 in LB (e.g. 500 μL overnight in 50 ml LB in a 250 ml flask). Incubate at 37°C with shaking to an OD600 of 0.375.
   Culture growth beyond OD 0.4 decreases transformation efficiency.
2. Aliquot 20 ml of the culture into chilled 50 ml tubes. Leave the tubes on ice for 5-10 minutes.
   Keep cells cold for all subsequent steps.
3. Centrifuge cells for 7 minutes at 1600 g, 4°C. Allow centrifuge to decelerate without brake.
4. Discard supernatant and resuspend each pellet in 4 ml ice cold CaCl2 solution.
5. Centrifuge cells for 5 minutes at 1100 g, 4°C.
6. Discard supernatant and resuspend each pellet in 4 ml ice cold CaCl2 solution. Keep on ice for 30 minutes.
7. Centrifuge cells for 5 minutes at 1100 g, 4°C.
8. Discard supernatant and resuspend each pellet in 800 μL ice cold CaCl2 solution.
   It is important to resuspend this pellet well.
9. Aliquot 100 μL of this suspension into microcentrifuge tubes. Freeze in liquid nitrogen and store at -80°C.

Transformation:

1. Thaw competent cells on ice for 10 minutes.
2. Add 1-3 μL prepped plasmid DNA to each 100 μL aliquot and incubate on ice for at least 30 minutes.
3. Transfer to 42°C for 2 minutes.
4. Put on ice for 5 minutes.
5. Add 900 μL of LB + 20 mM glucose or SOC and incubate at 37 °C with shaking or rotation for at least one hour.
6. Plate on selective media and incubate overnight at 37 °C.

Notes

• To prepare a greater amount of competent cells, inoculate four 100 ml cultures with 1 ml overnight culture each, make 50 ml aliquots, and resuspend at 10 ml, 10 ml, and 2 ml.