BIO-RAD电转化仪使用教程(自制)

BIO-RAD Gene Pulser Xcell Electroporation SystemBIO-RAD电转化仪使用教程（自制）cexoihtydx 20110902一电转仪示意图Figure 1 connecting the shockpad to the Gene Pulser Xcell main unit.Figure 2 Shockpod with cuvette.Figure 3 Gene Pulser Xcell front panel.二电转仪主界面在主界面中，我们经常会用到：4. Pre-set protocols和5. User protocolsPre-set protocols(预置方案)中，有Bacterial,Fungal和Mammalian三种预置方案。

下面简单介绍一下Bacterial中E. coli和Fungal中Pichia pastoris的电转化方案和注意事项。

三Electroporation of Bacterial Cells (E. coli)1 制备电转化感受态细胞1). Inoculate 5 ml of a fresh overnight E. coli culture into 500 ml of L-broth ina 2.8 L Fernbach flask.2). Grow the cells at 37°C shaking at 300 rpm to an OD600 of approximately 0.5–0.7. The best resultsare obtained with cells that are harvested at early- to mid-log phase; the appropriate cell densitydepends on the strain and growth conditions but should be about 4–5 x 107cells/ml.3). Chill the cells on ice for ~20 min. For all subsequent steps, keep the cells as close to 0°C as possi-ble (in an ice/water bath) and chill all containers in ice before adding cells. Transfer the cells to asterile, cold 500 ml centrifuge bottle and centrifuge at 4000 xg for 15 minutes at 4°C.4). Carefully pour off and discard the supernatant. It is better to sacrifice yield by pouring off a fewcells than to leave any supernatant behind.5). Gently resuspend the pellet in 500 ml of ice-cold 10% glycerol. Centrifuge at 4000 xg for 15 minutes at 4°C; carefully pour off and discard the supernatant.6). Resuspend the pellet in 250 ml of ice-cold 10% glycerol. Centrifuge at 4000 xg for 15 minutesat4°C; carefully pour off and discard the supernatant.7). Resuspend the pellet in ~20 ml of ice-cold 10% glycerol. Transfer to a 30 ml sterile Oakridge tube.Centrifuge at 4000 xg for 15 minutes at 4°C; carefully pour off and discard the supernatant.8). Resuspend the cell pellet in a final volume of 1–2 ml of ice-cold 10% glycerol. The cell concentration should be about 1–3 x 1010cells/ml.9). This suspension may be frozen in aliquots on dry ice and stored at -70°C. The cells are stable forat least 6 months under these conditions.2 电转化转化参数：Pre-set protocols (E. coli)V oltage(V) 1800Capacitance(µF) 25Resistance(ohm) 200Cuvette(mm) 11). Thaw the cells on ice. For each sample to be electroporated: place a 1.5 ml microfuge tube onice, place either a 0.1 or 0.2 cm electroporation cuvette on ice, and place a 17 x 100 mm tubewith 1 ml of SOC at room temperature.2). To a cold, 1.5 ml polypropylene microfuge tube, add 20 µl of cell suspension if electroporating in 0.1 cmcuvettes, or 20–40 µl of cell suspension if electroporating in 0.2 cm cuvettes. Add 1 to 2 µlof DNA(DNA should be in a low ionic strength buffer such as water or TE). Mix well and incubateon ice for ~1 minute. (Note: it is best to mix the plasmids and cells in a microfuge tube since the narrow gap ofthe cuvettes prevents uniform mixing.)3). From the Home screen on Gene Pulser Xcell open the Pre-set Protocols screen, then the BacterialProtocol screen (press 4, then Enter twice). When using the 0.1 cm cuvettes, press Enter to open E. coli, 1mm cuvette Protocol Detail screen. When using the 0.2 cm cuvettes, press 2 then Enter, or 3 then Enter, to select the Protocol Detail screens for E. coli to pulse at 2.5 or 3.0 kV, respectively.4). Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom. Place the cuvette in the ShockPod. Push the chamber lid down to close.5). Pulse once.6). Remove the cuvette from the chamber and immediately add 1 ml of SOC medium to the cuvette.Quickly but gently resuspend the cells with a Pasteur pipette. (The period between applying the pulse and transferring the cells to out growth medium is crucial for recovering E. coli transformants (Dower et al., 1988). Delaying this transfer by even 1 minute causes a 3-fold drop in transformation.This decline continues to a 20-fold drop by 10 minutes.)7). Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37°C for 1 hour,shaking at 225 rpm.8). Check and record the pulse parameters. The time constant should be close to 5 milliseconds. Thefield strength can be calculated as actual volts (kV) / cuvette gap (cm).9). Plate on LB plates with antibiotic.3 溶液和试剂1). L-Broth: 10 g Tryptone peptone, 5 g Yeast extract, 5 g NaCl; dissolve in 1.0 L water.Autoclave.2). LB agar plates with selective antibiotic: prepare L broth as above, adding 15g of agar perliter. Autoclave. Cool to 55–60°C and add antibiotic. Pour 12–15 ml per 100 mm plate.3). 10% (v/v) Glycerol: 12.6 g glycerol (density = 1.26 g/cc) in 90 ml of water. Autoclave or filtersterilize.4). TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTA.5). SOB: 2.0 g Tryptone peptone, 0.5 g Yeast extract, 0.2 ml 5 M NaCl, 0.25 ml 1 M KCl; dissolvein 90 ml water. Adjust pH to 7.0. Bring volume to 100 ml. Autoclave. Add 1.0 ml sterile 1 M MgCl2and 1.0 ml sterile 1 M MgSO4.6). SOC: to 100 ml SOB, add 2.0 ml sterile 1 M glucose (sterilize by filtration).4 注意事项1). 细菌电转化电压一般默认为1.8 KV即可，某些细菌可能会需要更高的电压，可以参考电转化仪器厂商的相关资料以及文献报道。