Chemically Competent Cells


 * 1) Pick a single bacterial colony and start a 5 ml culture in LB. There is no antibiotic resistance involved with these bacteria so sterile technique is important! 
 * 2) Grow overnight at 37 °.
 * 3) Put the rotor, 0.6 ml Eppendorf tubes and your reagents (MgCl2-CaCl2 solution, 0.1 M CaCl2+ 10% glycerol) in the cold room.
 * 4) Inoculate 200 mls LB with 2 mls overnight culture in a 2L flask and incubate at 37 °, monitoring the growth of the culture by taking its OD600.
 * 5) You will harvest the bacterial culture wen the OD600 reaches 0.35-0.4 and this may take several hours. If it is higher than this, you cannot go forward with the rest of the protocol.
 * 6) When your culture is getting close, put four 50 ml falcon tubes on ice and turn on the centrifuge; setting the temperature to 4 °.
 * 7) Transfer the culture to the cold 50 ml falcon tubes.
 * 8) Keep on ice for 10 minutes. At this point it is very important that the cells are kept ice cold for the remainder of the protocol. ALWAYS keep them on ice!
 * 9) Spin the cells down at 4000 RPM for 10 minutes at 4 °.
 * 10) Decant the media carefully and stand the tubes upside down for 1 minute to get out as much of the media as possible.
 * 11) Re-suspend each pellet by carefully swirling or gently vortexing in 15 mls of cold MgCl2-CaCl2 solution (60 mls total). Spin down the cells again.
 * 12) Decant the media carefully and stand the tubes upside down for 1 minute to get out as much of the media as possible.
 * 13) Re-suspend the pellet by carefully swirling or gently vortexing in 1 ml (4 mls total) ice cold 0.1 M CaCl2+ 10% glycerol.
 * 14) Aliquot 50 uL/0.6 ml tube in the cold room and freeze in liquid nitrogen. 
 * 15) Store at -80 °.