APPENDIX A

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): 70% ethanol; DH10B competent cells; vector/insert ligation reaction (see CHAPTER 5, CHAPTER 14, and CHAPTER 15); Gibco BRL Cell-Porator® Electroporation System (cat. no. 11609-013); Gibco BRL Cell-Porator® Voltage Booster (cat. no. 11612-017); disposable Cell-Porator® cuvettes

METHODS:

1. Fill the chamber safe with ice (FIGURE A.1). Turn on the power supply and the voltage booster. Make sure the voltage booster resistance is set on 4000 ohms (4 k). On the power supply, the capacitance should be set to 330 µF, the charge controller should be set to "Charge", the trigger speed should be set to "Fast", and the impedance toggle should be set on "Low ".
2. Thaw one tube of DH10B competent cells for every ten microliters of ligation reaction.
3. Make up a transformation reactions by mixing 2.0 µl of ligate with 20 µl of competent cells in a 0.65 ml microcentrifuge tube. Tap tubes gently to mix contents.
4. Using a pipettor equipped with a standard 200 µl tip, transfer the mixture from a tube to an electroporator cuvette. Place the mixture directly between the two electrode knobs of the cuvette (FIGURE A.2). Make sure that there are no bubbles in the ligate/bacterial solution. Close the cuvette.
5. Repeat step 5 until all transformation reactions are loaded into cuvettes.
6. Place the first four loaded cuvettes into the cuvette holder. Place the cuvette holder into the ice-filled chamber safe so that each cuvette is surrounded by ice (FIGURE A.3).
7. Close and lock the lid to the chamber safe. Insert the power-out plug into the plug inlet in the lid of the chamber safe (FIGURE A.4). Turn the cuvette dial on the chamber safe lid so that it is pointing to the number "1". The cuvette lying under the number "1" on the lid is ready for electroporation.
8. With the charge controller set on "CHARGE", push the "UP" button until a voltage of 330 appears in the LED display.
9. Flip the charge controller to the "ARM" position. Allow the voltage to drift down to approximately 325 v. Press the red "TRIGGER" button. The voltage will drop to zero (or thereabouts) as the bacteria in cuvette number one are exposed to a burst of electricity.
10. Flip the charge controller back to the "CHARGE" position. Turn the cuvette dial so that it is pointing to the number "2". Repeat steps 9-10 until all four cuvettes in the chamber safe have been shocked.

­ Note A.1: Be careful not to shock cells in a cuvette more than once.

11. Remove the power plug from the lid of the chamber safe. Unlock and open the chamber safe. Gently remove the cuvettes and place them on ice.