The following protocol routinely gives 10 exp9 transformants/ug via electroporation using pGEM1. The cells are aliquoted and stored at -80 C until needed. Typically I aliquot 60 ul of cells and transform with 0.5 ul of a 10:1 or 20:1 dilution of a ligation.
for 400 ml LB inoculate with 1/100 volume from fresh O/N (4 ml into 400 ml LB in 1-liter flask) grow to OD 0.6 chill cells on ice 15-30 min. pellet cells 0 deg C, 4000XG, 15 min, (5k RPM GSA rotor) Wash 2x: resuspend cells in 200 ml 1mM HEPES pH 7 10% Glycerol pellet as above resuspend to final volume of 2 ml in GYT (add 1.5 ml GYT) cell concentration should be 3x10exp10 cells/ml aliquot 40 ul to 70 ul freeze in dry ice ethanol GYT 50 ml 10% Glycerol 6.3 g (d = 1.26 g/ml) 0.125% Yeast Extract 62.5 mg 0.25% Tryptone 125 mg volume to 50 ml filter sterilize LB 400 ml 4 g Tryptone 2 g Yeast Extract 2 g NaCl 0.4 ml 1 N NaOH vol to 400 ml autoclave 20 min HEPES/Glycerol 400 ml 10% Glycerol 50.4 g (40 ml) 360 ml H20 autoclave cool add 0.4 ml 1M HEPES pH 7