How do I transform or electroporate E. coli cells with plasmid constructs?
Transformation: There are many methods which will give you a number of transformants when you only need to change hosts or if you recieve plasmid DNA from someone else. A simple technique is to centrifuge exponentially growing (~2 x 10^8 cfu/ml) cells at 3000 rpm and then resuspend them in 1:20 volume of cold (4 C) 75mM CaCl2. Generally, 50-100 ul of concentrated cells are placed in a microcentrifuge tube. Keep the cells on ice with DNA added, heat shock at 42 C for 90-120 seconds, add fresh broth to express for about an hour at 37 C. Plate the undiluted mixture or a microfuge concentrated portion directly onto dry, selective media. For “ultra-competent” E. coli cells, try the method of Inoue1990 which is the method preferred by netters: • Inoculate from an overnight grown in LB • Grow in 250 ml “SOB” at 18 degrees C until OD600 = 0.6 • On ice for 10 minutes. • Spin at 2500 x g (3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C. • Resuspend cells gently in 80 ml of ice cold “TB”
