电穿孔技术

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7.6 Universal 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：www.easylabs.com.cn 发布时间：2009-08-31 查看次数：1935   站长注：下文是发 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 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 electroporation Pulsed 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。 ProcedurePreparation of the cells 1. Collect the cells to be transfected from cultures in the mid- to late-logarithmic phase of growth. Use either a rubber policeman or trypsin to release adherent cells. Centrifuge at 500g at 4 °C for 5 min. 在对数生长期收集细胞，离心沉淀。 2. Resuspend the cell pellet in 0.5volume of the original growth medium and measure the cell number using a hemocytometer. 用培养基重悬细胞，计数 3. Collect the cells by centrifugation, as described in Step 1 and resuspend them in growth medium or phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4) at 15–25 °C at a concentration of 2.5 106 to 2.5 107 cells/ml. 离心收集细胞，重悬细胞于培养基或PBS中，调整细胞密度至2.5 106 to 2.5 107 cells/ml. 4. Transfer 400ul aliquots of the cell suspension (106–107 cells) into as many labeled electroporation cuvettes as needed. Place the loaded cuvettes on ice. 转移400ul细胞悬液至电转杯中，将之放在冰上。 5. Set the parameters on the electroporation device. (A typical capacitance value is 1,050 F.) Voltages range from 200 to 350 V, depending on the cell line, but generally average 260 V. Use an infinite internal resistance value. Discharge a blank cuvette containing PBS at least twice before electroporating cells. 设置电转参数，一般电容为1050 F，电压范围200到 350 V（因细胞不同而不同），一般260V。在电穿孔之前，需用含PBS的空电转杯通电两次。 Introduction of the DNA 6. Add 10–30 g of plasmid DNA in a volume of up to 40 l to each cuvette containing cells. (Some investigators add carrier DNA (for example, salmon sperm DNA) to bring the total amount of DNA to 120 g.) Gently mix the cells and DNA by pipetting the solution up and down. Proceed to Step 7 without delay.Do not introduce air bubbles into the suspension during the mixing step. 加入10–30 g质量DNA于含有细胞的电转杯中，不超过40ul，轻轻混匀后，立即进入下一步 7. Immediately transfer the cuvette to the electroporator and discharge the device. After 1–2 min, remove the cuvette, place it on ice and proceed immediately to the next step. 将电转杯放入电转仪中，通电。1-2分钟之后，取出放在冰上，立即进入下一步操作。 8. Transfer the electroporated cells to a 35-mm culture dish using a micropipettor equipped with a sterile tip. Rinse out the cuvette with a fresh aliquot of growth medium and add the washings to the culture dish. Transfer the dish to a humidified incubator at 37 °C with an atmosphere of 5–7% CO2. 将细胞转入培养皿中放入培养箱培养。 9. Repeat Steps 6–8 until all of the DNA cell samples have been treated. Recording the actual pulse time for each cuvette will facilitate comparisons between experiments. 重复6–8步，将各组细胞电转。 10. If the objective is stable transformation of the cells, proceed directly to Step 11. For transient expression, examine the cells 24–96 h after electroporation using an appropriate assay. 如果是要获得稳定转染细胞，直接进行第十一步。暂时转染，在电穿孔后24–96 h后检测。 11. To isolate stable transfectants, incubate for 48–72 h in complete medium, trypsinize the cells and replate them in the appropriate selective medium. Change the selective medium every 2–4 d for 2–3 weeks to remove the debris of dead cells and to allow colonies of resistant cells to grow. Thereafter, clone individual colonies and propagate for the appropriate assay. 要分离稳定转染的细胞，在完全培养基中培养48–72 h，胰酶消化后用选择培养基培养。SourceThis protocol was adapted from "DNA transfection by electroporation" in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.) 16.33–16.36 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001; ). REFERENCES 1. Wong, T.-K. & Neumann, E. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107, 584–587 (1982). | PubMed | ISI | ChemPort | 2. Potter, H. , Weir, L. & Leder, P. Enhancer-dependent expression of human immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. USA 81, 7161–7165 (1984). | PubMed | ChemPort |