Gene pulser

Resuspend 1-3 x 107 cells in 0.5 mL DMEM, and transfer them to a Gene Pulser cuvet Mix the DNA with the cells by gentle pipeting, and leave the cuvet on ice for 5 min... 

Set up a Gene Pulser to give a 2500 V pulse, using a 25 pF capacitor, and adjust the resistance to 200 Q. Place the cuvette into the electroporation chamber and pulse once. 

For electroporation ELECTROMAX DH10B competent cells with pUC119 control plasmid (Gibco-BRL). We use Gene Pulser (Bio-Rad, Richmond, CA) equipped with Pulse Controller and E. coli Pulser cuvet with 0.1-cm gap (Bio-Rad) for electroporation of E. coli. 

Electroporate at 270 V-500 pF using Gene Pulser equipped with Capacitance extender (Bio-Rad). Place the cuvet at room temperature for 10 min, transfer the cells to 30 mL RPMI/10% FCS in a 75-cm2 flask, and culture in a humidified 37°C, 5% C02 incubator. 

Electroporation apparatus (Bio-Rad Gene Pulser with Capacitance Extender, BTX ECM 600 or similar instrument). 

Add 250 pL cell suspension to each cuvet. Mix gently and proceed with the electroporation. The settings for electroporation with the Bio-Rad Gene Pulser are as follows 960 pF 220 V. The settings using the BTX ECM 600 are as follows 1200 pF Resistance Timer R5 220 V (Note 15). 

Gene Pulser (BioRad, Hercules, CA) or other electroporation device capable of... 

Electroporate with a Gene Pulser (BioRad), or other electroporator capable of delivering a similar pulse (2.5 kV, 25 pF, and 200fi) (see Note 13). 

Several electroporation devices were developed and are being used (Potter, 1988). In the protocols described here the devices used were the Gene Pulser from Bio-Rad (Cat. No. 165-2977) and a device constructed at EMBL (available on request). Electroporation cuvettes with 0.2-cm and 0.4-cm electrode gaps are from Bio-Rad (Cat. Nos. 165-2088 and 165-2086). 

F with EMBL device, 25 //F with Gene Pulser). 

Transfer DNA-bacteria mixture into precooled cuvette with 0.2-cm electrode distance. Make sure that the drop of solution is positioned between the electrodes at the bottom of the cuvette. Apply a single pulse with settings of 12.5 kV/cm, 25 /j,F, 200 D (Gene Pulser, Bio-Rad). 

Transfection by Electroporation. Materials 15 mg of plasmid DNA, 0.4 cm Electroporation cuvettes (BioRad, Hercules, CA, 165-2088), Electro-porator (BioRad Gene Pulser II) and Electroporation media (EM) (20% FBS/SF RPMI), 6-well tissue culture plates, SF RPMI, and complete RPMI. 

Human fibroblasts can be transfected by electroporation. For transfection, 4 X 10 cells are resuspended in 500 //I PBS containing 20 /itg DNA. The suspension is placed in an electroporation cuvette (Bio-Rad Laboratories, UK) with a gap of 0.4 cm, and kept in ice for 10 min before electroporation. For the electroporation, a Gene-Pulser Electroporator (Bio-Rad Laboratories, UK) can be used with the following working parameters voltage, 380 V capacitance, 850 fjF. After the pulse, the cells are resuspended in normal culture medium and plated onto cover-slips in 24-weU plates at a density of 1.5 x 10 cellsAvell (Bonazzi et al., 2005). 

Bacterial transformation with the ligation products can be performed in a variety of ways. We use the Gene Pulser II (Bio-Rad) which we find to be efficient, reproducible, and fast Transformations are performed using 1 pL of the ligation reactions and electro-competent JM109 E colt. Using 0.2-cm electrode-gap electroporation cuvets (Bio-Rad), the Gene Pulser II should be set at 200 SI resistance, 25 pFD capacitance and 2.5 kV. For more details on this method, see the manufacturer s directions... 

The Gene Pulser (Bio-Rad) provides a fast, efficient way to perform the numerous bactenal transformations that must be undertaken with the differential-display technique. We prepare our own electrocompetent bacteria from frozen stocks as suggested by the manufacturer... 

In an electroporation cuvet mix 25 pg of linearized-targetmg vector with 10 cells (0.8 mL of cell suspension) Sit for 5 min and then electroporate at 960 pF, 250 V in a Bio-Rad gene pulser with a capacitance extender see Note 11) Gently mix to disperse the pH gradient caused by the electroporation and sit 10-15 mm at room temperature. 

The EPI300 (Epicentre) electrocompetent E. coli cells work well with this procedure. Combine 3 pi DNA with 30 pi of these cells in a 1-mm cuvette (BioRad) and electroporate the cells at 1,200 V, 25 pF, and 200 Q using a Gene Pulser Xcell electroporation system (BioRad). 

Transfer into a 0.1-cm gap prechilled cuvette. Set the Gene Pulser at 1.8 kV, 25 pF capacitance, and 2000 resistance. Electroporate the DNA into the E. coli and immediately add 1 ml of SOC medium. Transfer the cells into a 15-ml falcon tube. 

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