Competent cells preparation

Cells have to be handled on ice and in a cold centrifuge.
Grow E. coli in LB media until OD600 reaches 0.5-0.6.
1 ml of cells is used for 1 transformation.
Centrifuge and suspend cells in 1 ml of “Na” buffer (5 mM Tris−HCl pH 8,0, 100 mM NaCl, 5 mM MgCl2).
Centrifuge and suspend cells in 1 ml of “Ca” buffer (5 mM Tris−HCl pH 8,0, 100 mM CaCl2, 5 mM MgCl2).
Keep cells on ice for 20 minutes. Centrifuge and suspend cells in 50 µl of “Ca” buffer. Cells are ready for transformation.
Competent cells can be stored at – 80 °C for prolonged periods: add 15% of glycerol to competent cells, fast-freeze in liquid nitrogen and transfer to – 80 °C freezer.