3. Decant the supernatant into waste beaker (this must be sterilized before being dumped down the drain). Resuspend the pellet in 10 ml of ice cold 0.1 M CaCl2. This is most easily done by resuspending in 1 ml, using the P1000 pipette and then adding 9.0 ml. Cut about 0.5 cm from the end of the blue tip before pulling E. coli through it, since the cells are fragile in this high calcium solution and may lyse if sheared. After they have been resuspended, centrifuge at 2,000 rpm for 10 min. (Sorvall HS-4 rotor).