Incubate 10ml of E.coli BL21(DE3) or DH5α o/n without plasmid (from –80°C stock). To do that, add 10mL of sterile LB media in a 50ml sterile falcon tube. With the use of a yellow micropipette, scratch the surface of the frozen E.coli from the -80°C freezer and toss it into the Falcon containing the media. This must be done fast, and the E.coli must not be outside the freezer more than 1 or 2 minutes (Use the termoblock from the -20°C to keep the E.coli stock frozen).

Incubate 40rpm

Re-inoculate in a new 10mL LB. Incubate until absorbance is 0.6-0.7 OD550 2h 0m 0s.

Centrifuge 4000rpm,4°C

Under the Laminar flux Cabin, throw the supernatant and resuspend in the same volume of CaCl2 100millimolar (mM)cold and sterile. Do not use vortex! 

Incubate in ice 0h 30m 0s. 

Centrifuge again 4000rpm,4°C

Resuspend in 1/10th of the previous volume of cold and sterile CaCl2 and 15% glycerol. Aliquot them in 100µL (using 1.5ml centrifuge tubes).

These aliquots can be used right away or can be stored in the -80°C freezer.