BIO-RAD电穿孔

Gene pulser xcell TM Electroporation system quick guide产品名称: Gene Pulser Xcell 电穿孔系统产品型号: Gene Pulser Xcell 电穿孔系统产品展商: 众磊（北京）生物科技发展有限公司驻沪办简单介绍Gene Pulser Xcell 电穿孔系统Gene Pulser Xcell 电穿孔系统的详细介绍【介绍】电穿孔是功能强大的将核酸、蛋白及其它分子导入多种细胞的高效技术。

通过高强度的电场作用，瞬时提高细胞膜的通透性，从而吸收周围介质中的外源分子。

这种技术可以将核苷酸、DNA 与RNA 、蛋白、糖类、染料及病毒颗粒等导入原核和真核细胞内。

电转化相对其它物理和化学转化方法，是一种有价值和有效的替代方法。

Gene Pulser Xcell 系统的设计，基于Bio-Rad 15 年来在电转化技术上经验积累，它提供指数波和方波波型选择、系统配置选择及友好的用户界面。

主要特点指数波和方波波型确保所有细胞类型（原核及真核）均可获得最佳的电转化效果Bio-Rad 专利的* PulseTrac 电路和电弧保护设计，确保可重复性并保护样品模块化设计可根据研究需要选择系统用户友好的数字化界面，具有直观的编程以控制所有参数，包括附属模块的参数包括人工操作、预设规程、用户规程、一个优化规程及其它先进功能等程序选择指数波或方波脉冲选择Gene Pulser Xcell 系统可产生指数波和方波波型，使你选择最适合你细胞的波型与规程。

指数波和方波均能有效地用于电转化及电融合。

电穿孔波型对不同类型细胞的转化效率有很重要的影响。

左，指数衰变脉冲。

当一个充电至电压为V 0 的电容器放电到细胞，加在细胞上的电压随时间以指数方式下降。

从起始电压下降到V0 / e 所需的时间称为为时间常数τ, 一种方便的脉冲时间表达方式。

右，方波脉冲。

放电到样品后截断电容器脉冲可产生方波脉冲。

Bio-Rad MicroPulser电穿孔仪的操作及维护规程1、功能介绍选择预设程序电穿孔仪预设了常用的微生物电击程序，包括5个细菌和5个真菌电击程序。

如下:按程序即被调出，按"Raise"和"Lower"键显示出不同的真菌转化程序。

电击的参数自动按显示的程序设定。

同样的，选择"Bacteria "程序相同。

选择手动模式A.改变电压按"Settings"键，当"Manual"旁的LED灯亮时，显示屏显示电压值（单位kV）。

按"Raise"和"Lower"键在kV to kV之间改变电压设置。

如果仪器刚刚打开，显示值为""。

B.截短脉冲当"Manual"旁的LED灯亮时，同时按"Raise"和"Lower"键LED屏显示“t —表示为脉冲选择了时间。

开机时的默认设置为标准的指数衰减脉冲，衰减过程并不被截短，显示为“一一”。

同时按"Raise"和"Lower"键后只松开"Lower"键，显示数字为截短的脉冲持续时间，单位为毫秒（ ms），从1毫秒开始以毫秒为增量一直到4毫秒。

限定脉冲时间在1-4毫秒之间。

同时按"Raise"和"Lower"键后只松开"Raise"键，可以调整脉冲时间到更短。

脉冲功能按"Pulse"键到电容充电至设定电压；"PLS"显示在显示屏上。

脉冲完成后会发出一声响，如果是一次设置好的多重脉冲则在整个脉冲过程中每次脉冲后发出一声响，"PLS" —直在显示屏上显示。

要想手动多重脉冲，则可以在一次脉冲完成发出一声响后，再次按"Pulse"键。

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。

MicroPulser 电穿孔仪操作手册2018 年12 月27 日1、介绍（1 ）基本原理MicroPulser 电穿孔仪用于细菌、酵母和其他众多微生物的电击转化，转化时，高压电脉冲作用于悬浮在小体积高阻介质中的样品。

本系统由一个脉冲发生器（pulse generator ）模块、一个电击腔（shocking chamber ）和一个装有电极的电击杯（cuvette ）组成。

样本放置于电击杯的电极之间。

MicroPulser 模块包含一个电容器，将电容器充电至高电压，然后模块将电容器中的电流放电到试管中的样品中。

MicroPulser 的电容放电电路产生具有指数衰减波形的电脉冲，如下图。

当电容器放电至样品时，跨越电极的电压迅速上升至最大电压（or 峰值电压，peak voltage ；也称为初始电压，Vo ），并随时间（t ）减小，如下式：其中τ=R · C，为时间常数，是脉冲长度的简便表达式。

R 为电路电阻，单位为ohms （欧姆）。

C 为电容，单位为microfarad （微法拉）。

根据方程1 ，τ是电压下降至峰值电压1/e （~37% ）的时间。

MicroPulser 的内部电路被设计以使E.coli 、酿酒酵母及其他许多微生物可以得到最佳电穿孔，最佳转化效率发生在大约5ms 的时间常数内。

这些电穿孔条件是通过使用10 微法拉电容器和将600 欧姆电阻与样品池并联以及将30 欧姆电阻与样品池串联来实现的。

除时间常数外，电场强度是另一个决定转化效率的重要参数。

电场强度E，是施加于电极间的电压，公式为：其中，V 为施加的电压，d 为电极间的距离，单位为cm 。

电场强度和细胞的尺寸（size ）决定了横贯每个细胞的电压降，正是电压降可能是电穿孔中电压效应的重要表现。

30 欧姆串联电阻的目的是在发生电弧的情况下保护设备电路。

在正常操作条件下，当样本在高电阻介质中，电阻不会影响施加在样本上的电压。

使用BIO-RAD电泳仪进行SDS-PAGE凝胶电泳的操作规范实验原理根据蛋白分子量亚基的不同而分离蛋白，在样品介质和丙烯酰胺凝胶中加入离子去污剂和强还原剂后，蛋白质亚基的电泳迁移速率主要取决于亚基分子量的大小。

实验所用仪器FR-200A全自动紫外与可见分析装置上海复日科技有限公司电泳仪 BIO-RAD公司TS-1型脱色摇床江苏海门市其林贝尔仪器制造有限公司实验用试剂低分子量蛋白Maker TAKARA4*上样缓冲 TAKARAPagn Blue protein staining solution Fermentas试剂的配制1.贮液的配制（1）凝胶储液取30g丙烯酰胺+0.8g甲叉双丙烯酰胺0.8g ,先用35ml双蒸水溶解，搅拌，直到溶液变成透明，再用双蒸水稀释至100ml，过滤。

棕色瓶4℃保存一个月。

（2）1 mol/l Tris-HCL (PH 8.8)12.1g Tris(三羟甲基氨基甲烷)溶解在80ml双蒸水中，用4mol/l盐酸调PH至8.8。

再用双蒸水稀释至100ml，保存在4℃冰箱。

（3）1.0 mol/l Tris-HCL (PH 6.8)6.06g Tris溶解在40ml双蒸水中，用用4mol/l盐酸调PH至6.8。

再用双蒸水稀释至50ml，保存在4℃冰箱。

（4）10%过硫酸铵（APS）0.1g过硫酸铵+1ml双蒸水。

使用前新鲜配制（5）Tris–甘氨酸电泳缓冲液的配制（25mmol/L Tris；250mmol/L甘氨酸(pH8.3)）30.3gTris+ 144.2g甘氨酸+ 10gSDS，双蒸水定容至1L。

每次使用时10倍稀释。

（6）样品缓冲液使用4*SDS-PAGE loading buffer(Takara公司)，上样缓冲与样品比例1:3混匀，之后煮沸5min。

2.凝胶的配制注：上表所标体积为配制两块胶的用量。

若配制一块或多块，可按比例减半或加倍。

具体步骤如下：1、样品制备：40 µL蛋白+5*上样缓冲液10 µL，煮沸5 min，冷却后放冰箱下层保存，备用2、制胶1）用专用的医用棉口罩将胶板擦拭干净，电泳槽清洗干净，组装模具。

Table of ContentsSection 1Introduction and Safety Information:The Gene Pulser XcellSystem (1)1.1General Safety Information (1)1.2Electrical Hazards (2)1.3Mechanical Hazards (2)1.4Other Safety Precautions (2)Section 2Unpacking and System Installation (3)2.1Unpacking the System Components (3)2.2Setting up the System (4)2.2.1Setting up the Gene Pulser Xcell Main Unit and Connectingthe ShockPod (Cat. #s 165-2660, 165-2661, 165-2662,165-2666) (4)2.2.2Connecting the PC Module to the Gene Pulser Xcell MainUnit (Cat. #s 165-2660, 165-2662, and 165-2668) (5)2.2.3Connecting the CE Module to the Gene Pulser Xcell MainUnit (Cat. #s 165-2660, 165-2661, and 165-2667) (6)2.2.4ShockPod (Cat. #s 165-2660, 165-2661, 165-2662, and165-2669) (6)Section 3Gene Pulser Xcell Operating Instructions (8)3.1Section Overview (8)3.2Front Panel and Home Screen (9)3.2.1Description of Keypad (9)3.2.2Home Screen (10)3.2.3Help Screens (11)3.3Manual Operation (12)3.3.1Manual Operation (Guide Guide) (12)3.3.2Electroporation using Exponential Decay Pulses (12)3.3.3Electroporation Specifying Time Constant (14)3.3.4Electroporation using Square Wave Pulses (15)3.3.5Results Screens (17)3.3.6Saving a Program from Manual Operation (19)3.3.6A Saving in a Location without a Named User Entry (20)3.3.6B Saving in a Location with a Named User Entry (20)3.4Pre-Set Protocols (21)3.4.1Using a Pre-set Protocol (Quick Guide) (21)3.4.2Electroporation using a Pre-Set Protocol (22)3.4.3Modifying Pre-Set Protocol Parameters (25)3.4.4Saving Changes to Pre-Set Protocols (25)3.5User Protocols (26)3.5.1Using a User Protocol (Quick Guide) (26)3.5.2Creating a New User Name (26)3.5.3Creating a New User Protocol (26)3.5.4Modifying a User Protocol (30)3.5.5Deleting a User Name and a User Protocol (31)3.5.6Renaming a User Name or a User Protocol (33)3.6Last Pulse (34)3.7Optimize Operation (34)3.8Data Management (36)3.9Measurements (39)3.9.1Sample Resistance Measurements (39)3.9.2Calibration and Measurement of Capacitors in theCE Module (40)3.10User Preferences (41)3.10.1Setting the Clock (41)3.10.2Adjusting the Screen Intensity (42)3.10.3Sleep Function Setting (42)3.11The Pulse Trac System (43)3.11.1Pulse Trac System Description (43)3.11.2Pulse Trac Diagnostic Algorithm (44)Section 4Overview of Electroporation Theory (44)4.1Exponential Decay Pulses (45)4.2Square Wave Pulses (45)Section 5Factors Affecting Electroporation:OptimizingElectroporation (48)5.1Cell Growth (48)5.2DNA (49)5.3Electroporation Media (49)5.4Temperature (50)Section 6Electroporation of Bacterial Cells (52)6.1Escherichia coli (52)6.1.1Preparation of Electrocompetent Cells (52)6.1.2Electroporation (53)6.1.3Solutions and Reagents (53)6.2Staphylococcus aureus (54)6.2.1Preparation of Electrocompetent Cells (54)6.2.2Electroporation (54)6.2.3Solutions and Reagents (55)6.3Agrobacterium tumefaciens (56)6.3.1Preparation of Electrocompetent Cells (56)6.3.2Electroporation (56)6.3.3Solutions and Reagents (57)6.4Bacillus cereus (57)6.4.1Preparation of Electrocompetent Cells (57)6.4.2Electroporation (57)6.4.3Solutions and Reagents (58)6.5Pseudomonas aeruginosa (58)6.5.1Preparation of Electrocompetent Cells (58)6.5.2Electroporation (59)6.5.3Solutions and Reagents (59)6.6Streptococcus pyogenes (60)6.6.1Preparation of Electrocompetent Cells (60)6.6.2Electroporation (60)6.6.3Solutions and Reagents (61)6.7Lactobacillus plantarum (61)6.7.1Preparation of Electrocompetent Cells (61)6.7.2Electroporation (61)6.7.3Solutions and Reagents (62)Section 7Electroporation of Fungal Cells (62)7.1Saccaromyces cerevisiae (62)7.1.1Preparation of Electrocompetent Cells (62)7.1.2Electroporation (63)7.1.3Solutions and Reagents (64)7.2Schizosaccharomyces pombe (64)7.2.1Preparation of Electrocompetent Cells (64)7.2.2Electroporation (65)7.2.3Solutions and Reagents (65)7.3Pichia pastoris (65)7.3.1Preparation of Electrocompetent Cells (65)7.3.2Electroporation (66)7.3.3Solutions and Reagents (66)7.4Candida albicans (67)7.4.1Preparation of Electrocompetent Cells (67)7.4.2Electroporation (67)7.4.3Solutions and Reagents (68)7.5Dictyostelium discoideum (68)7.5.1Preparation of Electrocompetent Cells (68)7.5.2Electroporation (69)7.5.3Solutions and Reagents (69)Section 8Mammalian Cells (70)8.1Preparation of Electrocompetent Cells (70)8.1.1Attached Cells (70)8.1.2Suspension Cells (70)8.2Electroporation (70)8.3Solutions and Reagents (71)Section 9References (72)Section 10Specifications and Product Information (75)10.1System Specifications (75)10.2Product Information (76)Section 1The Gene Pulser Xcell™Electroporation SystemThe Gene Pulser Xcell is a pulse generator that uses capacitors to produce controlled exponential or square wave electrical pulses for cell electroporation. The unit is capable of producing pulses of up to 3000 V on a high-voltage circuit, and up to 500 V on a low-voltage circuit. For generating pulses on the high voltage circuit, capacitors of 10, 15, and 25 µF present in the Gene Pulser Xcell main unit are used and generating pulses on the low-voltage circuit requires use of capacitors in the CE Module. Exponential decay (or capacitance discharge) and square wave pulses are the most commonly used types of electrical pulse. Anin-depth discussion of these two waveforms can be found in Section 4.The Gene Pulser Xcell is a modular system, comprising of a main unit and two accessory modules,the CE module and the PC module, in addition to the shocking chamber and a cuvette with incorporated electrodes. The CE Module is recommended for use with the Gene Pulser Xcell main unit for electroporation of most eukaryotic cells, including mammalian cells and plant protoplasts. The CE Module should only be used with low-resistance media (<1000 ohms). For exponential decay pulses, the CE Module provides a means of controlling the capacitance of the circuit by increasing the time constant of the pulse. For square wave pulses, the CE Module provides the large capacitor necessary for delivering a square wave pulse into low resistance media. This module contains a set of capacitors with a functional range between 50 and 3275 µF and selectable in 25 µF increments. For square wave pulses, the CE Module provides the large capacitance, 3275 µF, necessary for delivering a square wave pulse into low resistance media.The PC Module is recommended for the electroporation of bacteria and fungi using exponential decay, as well as in other applications where high-voltage pulses are applied to samples of small volume and high resistance. The PC Module selects resistance of 50 ohms 1000 ohms in 50-ohm increments. The unit is used to control the resistance of the circuit by placing resistors in parallel with the sample, thereby provid-ing a means of reducing the time constant of an exponential decay pulse. This provides an effective means of controlling the time constant when using high-resistance media but has little effect on the time constant when using low-resistance media. The PC Module greatly reduces the likelihood of an arc occurring at high voltage. It is not recommended that the PC module be used for square wave pulses due to the increase in droop of the pulse that can occur (see Section 4).Both the PC Module and CE Module have integral leads that connect to the main unit (see Section 2 for installation) and both units are controlled directly from the user interface on the front panel of the main unit.1.1General Safety InformationThis Bio-Rad instrument is designed and certified to meet the safety requirements of EN61010 and the EMC requirements of EN61326 (for Class A) and conforms to the “Class A” standards for electromagnetic emissions intended for laboratory equipment applications. This instrument is intended for laboratory application only. It is possible that emissions from this product may interfere with some sensitive appliances when placed nearby or in the same circuit as those appliances. The user should be aware of this potential and take appropriate measures to avoid interference.No part of the Gene Pulser Xcell system should be used if obvious external case damage has occurred or the electronic displays are not functioning as described in the manual. This instrument is only to be used with the components provided (or their authorized additions or replacements) including, but not limited to, supplied cables and ShockPod. The operating temperature range for the Gene Pulser Xcell system and its associated components is 0–35°C.1There are no user serviceable parts within the unit. The operator should make no attempt to open any case cover or defeat any safety interlock. This instrument must not be altered or modified in any way. Alteration of this instrument will•Void the manufacturer’s warranty•Void the IEC 1010 safety certification•Create a potential safety hazardBio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended or by modification of the instrument not performed by Bio-Rad or an authorized agent.1.2Electrical HazardsThe Gene Pulser Xcell produces voltages up to 3,000 volts and is capable of passing very high currents. When charged to maximum voltage, the instrument stores about 400 joules. A certain degree of respect is required for energy levels of this order. System safety features prevent operator access to the recessed input jacks and to the recessed electrode contacts inside the sample chamber. These mechanical interlocks should never be circumvented.The pulse button is active whenever the character space in the lower right corner is flashing. There is high voltage present whenever the pulse button is depressed and “Pulsing” is shown on the LCD display on the front of the instrument. Because of the built-in safety interlock in the ShockPod, no pulse is delivered to the cuvette when the ShockPod lid is opened. If the capacitor has been partially charged but not fired (for example, when the charging cycle has been interrupted before the pulse is delivered), some charge may remain on the internal capacitor. This charge will dissipate over 1–2 minutes. However, the user cannot make contact with any charged electrical components due to the system safety features.1.3Mechanical HazardsThe Gene Pulser Xcell contains a patented arc-protection circuit that dramatically reduces the incidence of arcing in the cuvette when high voltage is delivered into the sample. The unit incorporates a circuit that senses the beginning of an arc and diverts current from the sample within <10 µsec, preventing, or greatly reducing, mechanical, visual, and auditory phenomena at the ShockPod. Should an arc occur, the sample chamber is effective in containing these small discharges, but nonetheless we strongly recommend wearing safety glasses when using the instrument.1.4Other Safety PrecautionsAvoid spilling any liquids onto the apparatus. Use only a paper towel or a cloth wet with either water or alcohol to clean the outside surfaces of the Gene Pulser Xcell.Use only the Bio-Rad cables supplied with the Gene Pulser Xcell.Use the ShockPod only in the assembled condition. Do not attempt to circumvent the protection of the ShockPod or use it while disassembled.Verify the display segments periodically.Read the instruction manual before using the Gene Pulser Xcell Electroporation System. For technical assistance contact your local Bio-Rad office or, in the US, call technical services at 1-800-4BIORAD(1-800-424-6723).2Warning: The Gene Pulser Xcell generates, uses, and radiates radio frequency energy. If it is not used in accordance with the instructions given in this manual, it may cause interference with radio communications. The Gene Pulser Xcell has been tested and found to comply with the limits for Class A computing devices (pursuant to Subpart J of Part 15 of FCC Rules) which provide reasonable protection against such interference when operated in a commercial environment. Operation of this equipment in a residential area is likely to cause interference. In this case the user will be required, at their expense, to take whatever measure may be required to correct the interference.Section 2Unpacking and System InstallationThe Gene Pulser XCell™ can be purchased as three systems as well as component parts:165-2660Gene Pulser Xcell Total System for eukaryotic and microbial cells, 100–240 V, 50/60 Hz, exponential decay and square wave delivery, includes main unit, CE Module, PCModule, ShockPod, 15 sterile cuvettes (5 each of 0.1, 0.2, and 0.4 cm gap),instruction manual165-2661Gene Pulser Xcell Eukaryotic System, 100/240 V, 50/60 Hz, exponential decay (25–3,275 µF range) and square wave delivery, includes main unit, CE Module,ShockPod, 5 sterile cuvettes (0.4 cm gap), instruction manual165-2662Gene Pulser Xcell Microbial System, 100/240 V, 50/60 Hz, exponential decay and square wave delivery, includes main unit, PC Module, ShockPod, 10 sterile cuvettes(5 each of 0.1 and 0.2 cm gap), instruction manual165-2666Gene Pulser Xcell main unit, 100/240 V, 50/60 Hz165-2667Gene Pulser Xcell CE Module, 25–3,275 µF range controlled by main unit, includes integral leads, 5 sterile cuvettes (0.4 cm gap), instruction manual165-2668Gene Pulser Xcell PC Module, 50–1,000 ohm range controlled by main unit, includes integral leads, 10 sterile cuvettes (5 each of 0.1 and 0.2 cm gap)165-2669Gene Pulser Xcell ShockPod shocking chamber, includes integral leads for connection to Gene Pulser Xcell, Gene Pulser II, or MicroPulser2.1Unpacking the System ComponentsRemove all packing material and connect components on a flat, dry surface near an appropriate electrical outlet.Upon receiving your instrument, please check that all items listed were shipped. If any items are missing or damaged, contact your local Bio-Rad office.3Section 3Gene Pulser Xcell™Operating Instructions3.1 Section OverviewThis section describes the operation of the Gene Pulser Xcell. The following summarizes the organization of this section.Section 3.2 below describes the functions of the keys on the front panel, the Home screen on the LCD display, and the Help functions built into the Gene Pulser Xcell.•The keys on the front panel of the main unit control the Gene Pulser Xcell. Section 3.2.1 describes the uses of these keys.•The Home screen provides easy access to programs built into the Gene Pulser Xcell as well as a direct method of manually entering pulse parameters to electroporate a sample. Section 3.2.2 describes these programs.•On-screen help is built into the software of the Gene Pulser Xcell. This may be accessed from any screen as described in Section 3.2.3.The Gene Pulser Xcell has three modes of operation: manual operation, pre-set protocols, and user protocols.Section 3.3 describes the Manual mode, which may be used to rapidly program the parameters necessary for delivering either an exponential decay or a square wave pulse.•Section 3.3.2 describes delivering an exponential decay pulse.•Section 3.3.3 describes delivering an exponential decay pulse but specifying a time constant rather than a capacitance and resistance value.•Section 3.3.4 describes delivering a square wave pulse.•Section 3.3.6 explains how programmed settings may be saved as user protocols.Section 3.4 describes the Pre-set protocols in which the pulse parameters have been optimized for a number of commonly used bacterial and fungal species and mammalian cell lines.•Pre-set Protocols may be called up and used directly (Section 3.4.2) or may be modified prior to being used (Section 3.4.3).• A modified Pre-set Protocol may be saved as a User Protocol (Section 3.4.4).Section 3.5 describes a custom mode (User Protocols) in which users may store optimized pulse parameters that they use in their own work.•User Protocols may be created in any of four ways:•In the User Protocols menu as a new protocol (Section 3.5.3).•In the User Protocols menu as an edited (modified) program (Section 3.5.4)•In the Manual menu as a new protocol (Section 3.3.4).•In the Pre-set Protocol Menu as a modified protocol (Section 3.4.4).•User Protocols, once created and saved, may be called up and used directly like Pre-set Protocols (Section 3.5.1).3.2 Front Panel and Home Screen3.2.1 Description of the keypadSee Figure 3.1 for a view of the Gene Pulser Xcell front panel.Alpha-numeric keys This array of keys permits entering numbers and letters into the Gene PulserXcell. Pressing the Shift key toggles between alphabetic and numeric input. Totype an alphabetic character, press the Shift key to enter alpha mode, thenpress the key with the appropriate letter. To type an a, press the 2 key once; totype a b, press the 2 key twice; to type a c, press the 2 key three times. To usethe same key twice, for example to type a then b, advance the cursor usingthe Right Arrow Key. The firmware on Gene Pulser Xcell will automaticallychange between alpha and numeric input depending on the parameter beingentered. In Protocol screens and Directory screens where a two-digit entrymust be made, the second digit must be entered within 2 seconds of the firstentry, otherwise the screen will default to the single-digit entry.Home key Returns the user to the Home screen from anywhere in the program.Back key Returns the user one level back in hierarchy toward the Home screen.Help key Displays on-screen help text.Save key Saves User Names and User Protocols.Delete key Removes only the last entry in the field; also used to remove User Name andUser Protocol files.Clear key Removes the entire line of the field.Enter key Indicates that a choice has been made and moves the cursor to the nextlocation.Arrow keys The Up and Down Arrow keys move the cursor up or down one row at a time.Depending on the screen and location of the cursor, the Right and Left Arrowsmay (1) move the cursor right or left one space at a time, (2) toggle forwardand backward one screen when there are multiple screens for the samemenu, or (3) increase or decrease numerical input values.Pulse button:Results in discharging a pulse. During this time “Pulsing” is shown on the LCDdisplay. A tone sounds to indicate that the pulse has been delivered. Whenmultiple pulses are delivered, a tone sounds after the last pulse has beendelivered. The Pulse is discharged to the electrodes if the ShockPod isconnected and the lid is closed. Otherwise, it is discharged safely within theinstrument.3.3 Manual Operation3.3.1 Manual Operation (Quick Guide)•From the Home screen:•Press Enter to select exponential decay;•Press 2, then Enter to select exponential decay but specifying a time constant;•Press 3, then Enter to select square wave.•Use the Up and Down Arrow keys to scroll through the parameter value spaces on the screen.When a parameter value is highlighted, use the keypad to enter a value, then press Enter to accept that value.•When the necessary parameter values have been entered, the Pulse button on the Gene Pulser Xcell is active.•Press the Pulse button to electroporate the sample.•Press the Back key to return to the Protocol Detail screen and to deliver another pulse.3.3.2 Electroporation using Exponential Decay PulsesSee Section 4.1 for a discussion of electroporation using exponential decay pulses.•When the Home screen (Figure 3.2) is selected, the number 1, corresponding to “Exponential protocol” is highlighted as the default choice. Press Enter to view the Protocol Detail Screen. If the number 1 on the Home screen is not highlighted, press 1 or use the Up or Down Arrow keys to highlight “Exponential protocol”, then press Enter to select. The Protocol Detail screen appears (Figure 3.3).•The following combination of parameters may be entered:Capacitance + VoltageCapacitance + Voltage + ResistanceThe three variables may be selected in any order, however, the set voltage will determine whether the high voltage or the low voltage circuit is to be used and will limit the range of the capacitance as indicated in Table 3.1. If a value for the capacitance is chosen that outside the range of the system, this value will default to the closest allowable value.Specifying a resistance value requires that the PC Module be attached. This is always recommended with high resistance media (i.e., >600 ohm) such as water, sucrose, glycerol, sorbitol, or polyethylene glycol. The PC Module places a resistor in parallel with the sample to reduce the resistance of the circuit. In this way, the time constant of a high-resistance sample may be reduced and controlled.•When the necessary parameter values have been specified, a flashing “P” appears in the character space in the lower right corner of the LCD display indicating that the pulse button on the Gene Pulser Xcell is active and that a pulse may be delivered.•Press the Pulse button to deliver a pulse. When the Pulse button is depressed, the LCD display will blank then show “Pulsing”. Upon completion, a tone will sound and the pulse measurements will be displayed on the Protocol Results screen (see Figure 3.8, Section 3.3.5).•Use the Left and Right Arrow keys to toggle between the Protocol Results screen and the last Protocol Detail screen.•With the Protocol Detail screen on the LCD display another pulse can be delivered using the same pulse parameters. To change the pulse conditions, press Enter; the cursor appears in the voltage parameter value. The parameters may be changed as described above.•To save the pulse parameters, see Section 3.3.6.•To review previously delivered pulses, see Section 3.8.3.3.5 Results ScreensAfter delivering a pulse, the LCD displays the results on a Protocol Results screen. This screen shows the results in both graphic and tabular form. Figures 3.6, 3.7, and 3.8 show examples of the results from an exponential decay pulse, an exponential decay pulse in which the time constant was specified, and a square wave pulse, respectively.Results of the last 100 pulses as well as of the pulse parameters are stored in Gene Pulser Xcell memory and are accessible from the Data Management program (Section 3.8).3.4.2 Electroporation using Pre-set ProtocolsThere are nine Pre-set Bacterial Protocols, six Pre-set Fungal Protocols, and 12 Pre-set Mammalian Protocols. These protocols are pre-programmed with the optimal parameters for the given organism. Use the Pre-set Protocols as follows.•From the Home screen, press 4 or use the Up and Down Arrow keys to highlight “Pre-set Protocols”, then press Enter to select and to show the Pre-set Protocols screen (Figure 3.12).•Press 1–3, or use the Up and Down Arrow keys, to highlight Bacterial, Fungal, or Mammalian Pre-set Protocols, then press Enter to select.•Use the alpha-numeric keypad or the Up and Down Arrow keys to scroll through the list of names.For the Bacterial and Mammalian Pre-set Protocols, use the Right and Left Arrow keys to toggle between the two screens. When the number corresponding to the desired name is highlighted, press Enter to select and to view the Protocol Detail Screen showing the electroporation parameters for that protocol. A flashing “P” in the character space in the lower right corner of the LCD display indicates that the Pulse button is active.•For example, from the Pre-set Protocols screen, press 3 to highlight “Mammalian”, then press Enter to select and to bring up the first Pre-set Mammalian Protocols screen with the names of six pre-set mammalian protocols (Figure 3.13). Press the Right and Left Arrow keys to togglebetween the two Mammalian Pre-set Protocols screens. Use the alpha-numeric keypad or the Up and Down Arrow keys to scroll through the list of names. When the desired name on theMammalian Pre-set Protocols screen is highlighted,press Enter to select that protocol and toview the Protocol Detail Screen showing the electroporation parameters for that protocol. Forexample, from the Mammalian Pre-set Protocols screen, press 1, then Enter to bring up theProtocol Detail Screen for CHO cells in a 2 mm cuvette (Figure 3.14).•Press the Pulse button to deliver a pulse. When the Pulse button is depressed, the LCD display will blank then show “Pulsing”. Upon completion, a tone will sound and the pulse measurements will be displayed on the Protocol Results screen (see Section 3.3.5).•Use the Left and Right Arrow keys to toggle between the Protocol Results screen and the last Protocol Detail screen.•With the Protocol Detail screen on the LCD display another pulse can be delivered using the same pulse parameters. To change the pulse conditions, press Enter; the cursorappears in the voltage parameter value. The parameters may be changed as described in Section 3.4.3.•To review previously delivered pulses, see Section 3.8.3.4.3 Modifying Pre-set Protocol ParametersThe parameters for a Pre-set protocol may be changed as follows.•From the Protocol Detail screen, press the Up or Down Arrow keys to highlight the value for one of the parameter settings (voltage, capacitance, or resistance for exponential decay pulses;voltage or time constant for time constant mode; pulse length, voltage, number of pulses, or pulse interval for square wave pulses). (Note: the waveform cannot be changed in the Pre-set Protocols Mode.) When the desired parameter is selected, use the alpha-numeric keypad to input the new value.Alternatively, use the Right and Left Arrow keys to incrementally increase or decrease, respectively, the parameter value. Use the Delete or Clear keys to correct entries. When the correct value has been specified, press Enter. If a value outside the limits of the Gene Pulser Xcell is selected, the value in the field will default to the closest permitted value. Use the Up and Down Arrow keys to select other parameter values to be changed, then use the alpha-numeric keypad or the Left and Right Arrow keys to enter the desired value.• A pulse may be delivered when appropriate parameters have been entered in the Protocol Detail screen and the character space at the lower right of the LCD display is flashing “P”.•To return to the last Protocol Detail screen, press the Back key or the Left Arrow key. Another pulse may be delivered using the same parameters shown on the LCD display. To return to the Protocol Results Screen, press the Right Arrow key. (Note: Returning to the Protocol Detail Screen returns to the modified parameters. To return to the Pre-set Protocol, press the Back key again to return to the Pre-set Protocols screen. This will remove any changes made.)•To change the pulse conditions, with the Protocol Detail screen on the LCD display, press Enter;the cursor appears in the voltage parameter value. The parameters may be changed as described above.•To review previously delivered pulses, see Section 3.8.3.4.4 Saving Changes to Pre-set ProtocolsChanges to a Pre-set Protocol may be saved as a User Protocol as follows:•Change the Pre-set Protocol as described in Section 3.4.3.•With the Protocol Detail screen open, press Save.•The first User Directory screen will appear (Figure 3.9); the second line will read “Choose location for protocol”.•Use the Right and Left Arrow keys to toggle between the two User Directory screens. Press 1–12 or use the Up and Down Arrow keys to highlight the User Name under which to store the protocol.Press Enter to select the User Name. The User Protocols screen will appear (Figure 3.10); the second line will read “Choose location for protocol”. If it is necessary to create a new User Name, seeSection 3.5.2.•Use the Right and Left Arrow keys to toggle between the two User Protocols screens. Press 1–12 or use the Up and Down Arrow keys to highlight a location for the new protocol. A protocol may be stored in a position without an entry (see Section 3.3.6A) or in a position with an entry (seeSection 3.3.6B). If necessary, delete a User Protocol as described in Section 3.5.5.•To use the saved protocol, press Enter to view the Protocol Detail screen. Press the Pulse button to deliver a pulse.。

Bio-rad 双向电泳系统标准操作规程1、目的：正确使用Bio-rad 双向电泳系统，确保Bio-rad 双向电泳系统正常运行。

2、适用范围：Bio-rad 7cm/11cm/17cm双向电泳系统。

3、责任人：双向电泳系统操作人员。

4、程序：4.1、第一向等电聚焦4.1.1、准备工作及注意事项用标配的刷子小心将聚焦盘清洗干净，注意聚焦盘两端的两根电极丝，晾干后备用；根据样品使用合适的水化上样液，对于不同的聚焦盘的上样体积可参考下表：4.1.2、上样4.1.2.1、从冰箱中取-20℃冷冻保存的水化上样缓冲液（不含DTT，不含Bio-Lyte）一小管，置室温溶解，加入合适的DTT与Bio-Lyte，充分混匀。

4.1.2.2、从小管中取出适量水化上样缓冲液与样品充分混匀。

4.1.2.3、取出-20℃冷冻保存的IPG预制胶条，室温中放置10分钟。

4.1.2.4、沿着聚焦盘或水化盘中槽的边缘至左而右线性加入样品。

在槽两端各1cm左右不要加样，中间的样品液一定要连贯。

注意：不要产生气泡。

否则影响到胶条中蛋白质的分布。

4.1.2.5、当所有的蛋白质样品都已经加入到聚焦盘或水化盘中后，用镊子轻轻的去除预制IPG胶条上的保护层。

4.1.2.6、分清胶条的正负极，轻轻地将IPG胶条胶面朝下置于聚焦盘或水化盘中样品溶液上，使得胶条的正极（标有+）对应于聚焦盘的正极。

确保胶条与电极紧密接触。

不要使样品溶液弄到胶条背面的塑料支撑膜上，因为这些溶液不会被胶条吸收。

同样还要注意不使胶条下面的溶液产生气泡。

如果已经产生气泡，用镊子轻轻地提起胶条的一端，上下移动胶条，直到气泡被赶到胶条以外。

4.1.2.7、在每根胶条上覆盖1-3ml矿物油，防止胶条水化过程中液体的蒸发。

需缓慢的加入矿物油，沿着胶条，使矿物油一滴一滴慢慢加在塑料支撑膜上。

4.1.2.8、对好正、负极，盖上盖子。

设置等电聚焦程序。

4.1.3、设置程序4.1.3.1、打开电源；4.1.3.2、根据情况选择水化（REHYDRATION），预设的程序（PRESET METHOD），储存的程序（STORED METHOD），新的程序（NEW METHOD）；4.1.3.3、如果只需要水化，选择水化（REHYDRATION）选项，在接下来的界面选择主动水化或者被动水化、水化温度、水化时间；4.1.3.4、如果需要跑完整的程序，选择新的程序（NEW METHOD）, 在接下来的界面选择是否水化，并设置相应的等电聚焦程序，设置完成后，在最后的界面选择总的胶条数、限电流和聚焦温度，然后开始运行程序。

电穿孔技术电穿孔缓冲液：配方1：200mM/L葡萄糖,5mM硫酸镁2mM/L疏基乙醇20mM/LTris-HCI ,pH值7.6Universal Electroporation Solution通用电转染缓冲液性能特点：1. 显著提高转染效率和细胞存活率2. 适用于难转染细胞等几乎所有细胞类型3. 与所有电融合/电穿孔仪兼容产品货号：UES0001产品规格：1ml保存：Universal Electroporation Solution保存于4度建议-20度长期保存保质期：正确的使用和保存条件下，保质期为6个月电转染次数：1ml Universal Electroporation Solution用0.4cm电极杯可进行5组实验用0.2cm电极杯可进行10组实验电转染条件：电压260V电容950μF操作步骤：1．收集细胞：用胰酶消化细胞，将细胞培养液吸入到15ml离心管中，1000rpm,5min，弃其上清。

2．用不含血清的培养液洗涤一次，弃其上清。

3．取2～10ug质粒加入到100ul电转缓冲液中，充分混匀。

4．用100ul混有质粒的电转缓冲液充分溶解细胞，转入0.2cm电转导入杯中。

5．将电转导入杯放入样品槽中，释放电脉冲，电击参数按电容1000 mF，电压150-500 V，间隔20V取值，取得最佳效果。

6．立即取出电击杯，分别用一次性吸管将混和液转移至加有完全培养基的一次性细胞培养瓶中，放入细胞培养箱中培养。

电穿孔法转染哺乳动物细胞来源： 发布时间：2009-08-31 查看次数：1935站长注：下文是发表于Nature Methods中的一篇关于电穿孔转染方法的文章，站长对其作了注释，方便大家理解。

电穿孔转染理论上可转染所有的组织细胞，因此对其他如脂质体、磷酸钙沉淀等方法转染效果不明显的细胞可选用此方法。

电转过程中，最重要的就是电穿孔仪的电压、电容以及与电泳缓冲液的选择。

提到电转仪，最出名的恐怕就属BIO-RAD了，他在1986年推出了世界上第一台电穿孔仪，并发布了多种细胞仪电转过程中的电压，电容，电转缓冲液等可省却大家很多的摸索过程，具体资料可到其主页上查找相关Nature Methods 3, 67 - 68 (2006)Transfection of mammalian cells by electroporationPulsed electrical fields can be used to introduce DNA into a wide variety of animal cells1, 2. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate–DNA coprecipitation. But as with other transfection methods, the optimal conditions for electroporation of untested cell lines must be determined experimentally.电穿孔转染可用于磷酸钙转染效率低下的细胞，具有操作简便，转染效率高等优点，但对于不同的细胞，其转染条件有很大的不同，需要进行摸索。

Electroporation1 原理细胞电穿孔的研究始于70年代。

所谓电穿孔(Electroporation)即是在外加短时强电脉冲时,在细胞膜双脂层上形成瞬时微孔,使细胞膜的通透性增强,离子,亲水分子,病毒颗粒,DNA,蛋白质以及染料颗粒等正常情况下不能通过细胞膜的分子得以进出细胞的一个生物物理过程。

目前,电穿孔技术已广泛用于生物技术,基因工程,临床医学等许多领域。

利用电穿孔可实现基因转染,细胞融合,以及将外源蛋白质分子插入细胞膜或导入细胞,激活细胞膜的传输因子,提高酶的活性,并可提高药物进入细胞的能力,增强癌症的化疗效果。

参考*低强度瞬态电磁脉冲对细胞膜的电穿孔效应及机理研究*2 应用Classic Applications for molecular delivery using electroporation include:Transformation of bacteria, yeast, and plantsTransfection of mammalian cellsElectroincorporation & Electroinsertion of proteinsSmall Molecule Delivery (RNA & oligos (knockouts), peptides & amino acids (proteomics)) Plant Hybrid & Hybridoma (monoclonal) productionEmerging Applications include:Adherent Cell Electroporation in situ (ACE & ACT)High Throughput Electroporation (HTE & HTC)Large Volume Processing (LVP)Electroincorporation & Electroinsertion of proteins (proteomics)in vivo Gene Delivery & In vivo Gene Therapyin ovo Gene Delivery (IOGD) and In & ex utero Gene Delivery (IUGD & EUGD)Cell Fusion & Nuclear Transfersource from:http://www.bioflow.co.kr/5 电转染常见细胞系,多为悬浮细胞African Green Monkey Kidney CV-1Embryonic Stem Cells (D3 cells)Rat Pituitary Tumar (GH3)Human Fibroblasts (GM0847)Human Cervical Carcinoma cells (HeLa)Human hepatocellular carcinoma Hep G2Primary Human Fetal Fibroblasts IMR-90Human Hematopoietic K562 CellsJurkat cellsL Cell L929LBRM cells (mouse T-cell lymphoma)Normal human melanocytesMouse Embryo Fibroblasts NIH3T3Primary Human Peripheral Blood LymphocytesPrimary Human Peripheral Blood Lymphocytes CCRF-CEM Tlymphoblastoid cells Raji Burkitt's lymphomaWEHI-3B Murine Myelomonocytic LeukemiaNamalwa Human Lymphoblastoid cellsCos 7 cellsHuman Neuroblastoma TP410NRat Cardiac MyocyteFibroblast NIH3T3 MouseHuman Melanoma (SKMe125)Hepatoma CellsMouse MacrophageMurine Fibroblast / C3H strainMurine T-LymphomaSmooth muscle HumanHuman 293 (Embryonic kidney cell)Breast Carcinoma MCF-7Mouse Melanoma (B16BL6)BHKB-LymphocyteProstate Tumor Cells LNCapRat Anterior Pituitary cellPC12COS 1Human Neuroblastoma SH-SY5YHuman FibroblastRat HepatocyteCOS 7 (Coaxial Electrode P/N 491)CR Mouse Skeletal MuscleT2 Cells (Hybrid of B & T Cells)Mouse Adrenal Carcinoma CellsRat Adipose CellsMyeloid Progenitor Cell (M07e)Bovine Aortic Endothelial CellsPrimary T CellsJurkatA549 Epithelial cells (human carcinoma tissue)NRK 52E Rat Kidney CellsNCI-H1299 Noon Small Cell Lung CancerHuman MyoblastK562 Human Chronic Myeloid LeukemiaCD 34+ Progenitor-derived Langerhans cellsCD 34+ Progenitor-derived Dendritic cellsMonocyte-derived Dendritic CellsMurine T-cellsHuman T-cellsHuman B lymphocytesDrosophila embryosPancreatic CellsHuman Bronchial Epithelial CellsPC12Rat Glioma C6Human prostatic stromal cellsPC3T24 Human Bladder CarcinomaMonocyte-derived Dendritic CellsHuman Dendritic cellsHippocampal neuron-HN2 cells Squamous cell carcinomaA549Human aortic smooth muscle cellsBone marrowMesenchymal cellsPrimary Neural Culture (small granular cells) Primary neural cell culture (Astrocytes) Chronic Lymphocytic Leukemia (CLL) source from*附有具体protocol及相关文献*: //附上图片一张电镜图片，看穿孔瞬间胞浆喷出。

MicroPulser操作方法1. 选择预设的程序设置MicroPulser预设了数种常见的微生物的电击程序如下:按"Settings"键选择"Bacteria", "Fungi",和"Manual "设置.当"Fungi "旁的LED灯亮时，预存的真菌转化程序即被调出，按"Raise" 和"Lower"键显示出不同的真菌转化程序。

电击的参数自动按显示的程序设定。

同样的，选择"Bacteria "程序相同。

2. 使用Micropulser的手动模式A. 改变电压按"Settings" 键，当"Manual".旁的LED灯亮时，显示屏显示电压值(单位kV). 按"Raise" 和"Lower"键在0.20 kV to 3.00 kV.之间改变电压设置。

如果仪器刚刚打开，显示值为"0.00"B. 截短脉冲.当"Manual".旁的LED灯亮时，同时按"Raise" 和"Lower"键LED屏显示“t—”，表示为脉冲选择了时间。

开机时的默认设置为标准的指数衰减脉冲，衰减过程并不被截短，显示为“——”。

同时按"Raise" 和"Lower"键后只松开"Lower"键，显示数字为截短的脉冲持续时间，单位为毫秒，从1毫秒开始以0.1毫秒为增量一直到4毫秒。

限定脉冲时间在1-4毫秒之间。

同时按"Raise" 和"Lower"键后只松开"Raise"键，可以调整脉冲时间到更短。

3. 脉冲功能按"Pulse"键到电容充电至设定电压；"PLS"显示在显示屏上。

中国民航大学硕士学位论文细胞电穿孔技术的研究姓名：孙云申请学位级别：硕士专业：通信与信息系统指导教师：徐宝强20070220中Ｎ民航人学硕卜学位论叟：物增敏、临床医学等诸多方面，给医疗技术带来了一场革命。

２１世纪是生物工程的时代，电穿孔技术也在不断发展完善，在应用领域出现了方兴未艾的局面，有着巨大的应用潜力。

因此分析研究细胞电穿孔的机理，研制我国具有自主知识产权的电转基因仪器和与之配套的多种电极，将具有重大意义，不仅会推动我国的生物工程的发展，而且必将产生良好的经济和社会效益。

１．２国内外研究现状１．２．１细胞膜结构细胞膜的结构非常复杂，主要是由脂质和蛋白质两类物质组成的复杂的超分子动态系统。

１９７２年Ｓｉｎｇｅｒ和Ｎｉｃｏｌｓｏｎ提出的液态镶嵌模型１２４｜，目前已得到人们的承认。

液态镶嵌模型认为细胞膜是一种可流动的、嵌有蛋白质的脂类双分子层结构。

嵌入其中的蛋白质具有多种功能，如离子通道、受体及各种酶等。

构成细胞膜的脂类主要有甘油磷脂、鞘磷脂、类固醇和糖脂，如图１－１所示。

这些脂类除了类固醇外，都有亲水的极性的头部和疏水的非极性的尾部。

脂类分子极性头部向外，疏水的尾部在内，构成细胞膜的基质——脂双分子层。

图１－１细胞膜的生物结构１．２．２细胞膜电穿孔模型对于细胞膜的可逆电穿孔，众多的理论模型都没有对电穿孔过程进行系统的描述，只有ＪａｍｅｓＷｅａｖｅｒ建立的瞬叁亲水孔模型１２５１，对电穿孔进行了宏观描述。

定性地说，电穿孔现象是由电能（因跨膜电位提高而产生的决定性能量）和“ＫＴ能量”（因热波动而产生的随机性能量）共同作用而引起的。

大量的观察发现，电穿孔图３－５组合电容３．１．５其他方面为了实现产业化，对原有的放电小池进行彻底改造，先后设计研制出几套模具，实现了ｌｍｍ、２ｒａｍ、４ｍｍ三种不同规格的放电小池。

其中２ｒａｍ、４ｍｍ小池的电极有粘贴型的，１ｌｎＩｎ、２ｒａｍ小池电极有采用工字铝注塑型的，如图３－６所示。

Bio-Rad成像系统使用规程1.1 打开电源：
打开机身背部左下方机身电源开关（左），后打开CCD电源开关（右）；
1.2 放入凝胶、调节位置
打开抽屉放入琼脂糖凝胶（左）或SDS-PAGE凝胶或PVDF膜（右）
打开Quantity One软件，点击“File”选择“ChemiDoc XRS”，按下“Select”键，选择“White EPI”，按下Live Focus按钮，勾选“Light on”，调节凝胶位置居于视野中央。

1.3 曝光
选择光源：
点击Iris、Zoom、Focus调节图像大小和最佳曝光参数
点击曝光按钮选项获得图像；
1.4 点击“File”中的“save”保存文件；
1.5 用完后关闭电源和清洁凝胶载物台；
●不准使用化学发光凝胶成像仪做切胶回收;
●使用前预热30min;
●使用完毕后请关闭电源和清洁凝胶载物台;。

Bio-rad Gene Pulser Xcell电穿孔系统一Gene Pulser Xcell电穿孔系统介绍电穿孔的过程中可以有2种波型来控制外界电压的变化，Gene Pulser Xcell可以提供2种波型，即指数波和方波。

1.Exponential Decay Wave（指数波）所谓指数波的方式，就是将已经充电到指定电压的电容进行快速的放电，放电的方式是以指数的形式进行的（如上面的slide显示）。

该种方式的电穿孔取决于2个参数，电场强度(kV/cm)和时间常数(ms)。

在实验过程中，采用具体的电击杯调整电压即可调整不同的电场强度。

此外，电容和电阻的具体值也可以在Gene Pulser Xcell的界面上进行设置。

•电容器充电到预定的电压(V0)放电后，即向样品释放脉冲，细胞表面的电压随时间按指数方式下降的•电容器的电压值达到峰值释放脉冲后迅速的衰减•电场强度 E (V/cm) 是用来描述电击杯外界电场环境的一个参数（E=V/d ）•脉冲时间是一般用时间常数来进行衡量(~37% of V0，V0/e)•脉冲的时间是由电阻和电容的大小所决定的备注：PC module作用PC（Parallel Capacitor）模块的本质就是在放电回路中在样品上并联一套电阻。

在放电回路中设置PC模块的目的：如果实验样品具有很高的电阻值，那么脉冲就需要维持相当长的时间，这样对细胞是会有损伤的，长时间的电场作用会导致细胞的裂解。

并联了PC模块后对样品而言电击是的电压并没有变化，但是PC 模块起到了分流的作用，可以使得脉冲的时间常数大大减小，可有效的维持细胞的活性。

并联了PC模块后，时间常数取决于该模块的电阻，电阻越小，那么放电的时间也就越短。

2. Square Wave（方波）对于某些细胞而言，采用指数波进行实验特别容易导致细胞的死亡。

对于这种细胞而言，采用方波的形式来进行电穿孔既可以进行有效的转染，又可以很好的保护细胞，维持细胞的活性。