BIO-RAD电转化仪使用教程(自制).pdfVIP

BIO-RAD Gene Pulser Xcell Electroporation System BIO-RAD 电转化仪使用教程 （自制） cexoihtydx 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 protocols Pre-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 in 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 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