Preparation of Electro-competent DH5 Cells
1. Prepare fresh patch of cells on LB from permanent (not from plate).
2. Grow in 5 ml 2X YT at 37°C overnight. Can use YENB (0.75% YE + 0.75% Nutrient Broth).
1. Inoculate 500 ml of 2X YT with 5 ml of fresh overnight culture. Grow at 37°C in a Fernbach flask
with vigorous shaking (225 rpm). Check OD600 until it reaches 0.5 - 0.75 (about 2-3 hours).
2. Transfer cells to two 250 ml-bottles. Chill cells on ice for 15 minutes or more.
3. Harvest cells by centrifugation in GSA rotor at 5,000 rpm at 4°C for 10 minutes.
4. Pour off all supernatant. Resuspend each pellet in 40 mls ice cold, sterile ddH2O. Transfer to
sterile 50 ml red cap tube. Spin at 2500 rpm in IEC at 4°C for 15 minutes.
5. Aspirate off all supernatant. Wash cell pellet once again in 50 mls ice cold, sterile ddH2O.
6. Aspirate off all supernatant. Resuspend each pellet in 20 mls ice cold, sterile 10% glycerol. Pool
into one 50 ml red cap tube. Spin at 2500 rpm in IEC at 4°C for 15 minutes.
7. Resuspend cells with 1 ml ice cold, sterile 10% glycerol (about 2 mls of competent cells).
8. Aliquot 75 µl cells per 0.5 ml microfuge tube. Freeze by placing tubes in powdered dry ice in ice
bucket. After frozen, store aliquots at -80°C. Thaw only once and use.
Transformation of DH5 by Electroporation
1. Precool on ice: 1. Sterile 0.2 cm gap electroporation cuvettes (or use 0.1 cm gap cuvette)