BIO-RAD Gene Pulr Xcell Electroporation System
BIO-RAD电转化仪使用教程（自制）population
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一电转仪示意图
Figure 1 connecting the shockpad to the Gene Pulr Xcell main unit.
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Figure 2 Shockpod with cuvette.
Figure 3 Gene Pulr Xcell front panel.
二电转仪主界面
在主界面中，我们经常会用到：
4. Pre-t protocols和
5. Ur protocols
Pre-t protocols(预置方案)中，有Bacterial,Fungal和Mammalian三种预置方案。
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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。writings on the wall
三Electroporation of Bacterial Cells (E. coli)17 year old
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 in
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a 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 pha; 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 subquent steps, keep the cells as clo 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.ms
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 the conditions.slow的副词
2 电转化
转化参数：
Pre-t protocols (E. coli)
V oltage(V) 1800
Capacitance(µF) 25
Resistance(ohm) 200
Cuvette(mm) 1
1). 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.黄鹤楼送孟浩然之广陵李白
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 Pulr Xcell open the Pre-t 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 lect the Protocol Detail screens for E. coli to pul 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 clo.
5). Pul 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 pul 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 caus a 3-fold drop in transformation.This decline continues to a 20-fold drop by 10 minutes.)
