发布时间 : 星期一 文章Bio-rad MicroPulser电穿孔仪中文说明书更新完毕开始阅读d712dc9cfd4ffe4733687e21af45b307e871f9ac
预冷的1M山梨醇。若用抗性筛选方法，则立即加入1ml冰预冷的YPD/山梨醇。轻柔转移细胞悬液至一个无菌管中。
? 检查和记录脉冲参数。时间常数应接近5ms。电场强度可以通过实际电压（kV）
/电击杯间隙宽度（cm）获得。
? 用营养缺陷突变株进行互补筛选时，细胞应立即涂布至含有1M山梨醇的限定琼脂
平板（minimal agar plate）上。当用抗性筛选方法时，电转后的细胞在30℃振荡培养1-2小时；在含有1M山梨醇的YPD琼脂平板上，用适当的抗生素进行电穿孔细胞的培养。30℃培养72-96小时。
5.3 电转化所需溶液和试剂
? YPD/HEPES：100ml YPD培养基，20ml 1M HEPES，pH8.0。 ? 1M DTT：1.55g DTT，溶于8ml水，定容至10ml，过滤除菌。
? YPD/山梨醇：10g yeast extract，20g peptone，182.2g山梨醇，溶于700ml
水，定容至900ml。高压灭菌。临用前加入100ml无菌20%葡萄糖。
References
1. Allen, S.P. and Blaschek, H.P., Factors involved in the electroporation- induced
transformation of Clostridium perfringens, Appl. Environ. Microbiol., 54, 2322 (1990).
2. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.,
and Struhl, K.(eds.) Current Protocols in Molecular Biology, John Wiley & Sons, NY (1987).
3. Becker, D.M. and Guarente, L., High-efficiency transformation of yeast by
electroporation, Methods Enzymol., 194, 182 (1991).
4. B?ttger, E. C., High-efficiency generation of plasmid cDNA libraries using
electro-transformation, BioTechniques, 6, 878 (1988).
5. Chang, D.C., Chassy, B.M., Saunders, J.A., and Sowers, A.E. (eds.) Guide to
Electroporation and Electrofusion, Academic Press, Inc., San Diego (1992). 6. Cregg, J.M. and Russell, K.A., Transformation, in Methods in Molecular Biology,
103, Higgins, D.R. and Cregg, J.M. (eds.), Humana Press, Totowa, NJ, 27 (1998).
7. Cruz-Rodz, A.L. and Gilmore, M.S., High efficiency introduction of plasmid DNA
into glycine treated Enterococcus faecalis by electroporation, Mol. Gen. Genet., 224, 152 (1990).
8. Dennis, J.J. and Sokol, P.A., Electrotransformation of Pseudomonas, in Methods
in Molecular Biology, 47, Nickoloff, J.A., ed., Humana Press, Totowa, NJ, 125 (1995).
9. Dower, W. J., Electroporation of bacteria: a general approach to genetic
transformation, in Genetic Engineering—Principles and Methods, 12, Plenum Publishing Corp., NY, 275 (1990).
10. Dower, W. J., Miller, J. F., and Ragsdale, C. W., High efficiency transformation of
E. coli by high voltage electroporation, Nuc. Acids Res., 16, 6127 (1988). 11. Dower, W.J., Chassy, B.M., Trevors, J.T., and Blaschek, H.P., Protocols for the
transformation of bacteria by electroporation, in Guide to Electroporation and Electrofusion, Chang, D. C., Chassy, B. M., Saunders, J. A., and Sowers, A. E. (eds.), Academic Press Inc., 485, San Diego (1992).
12. Heery, D. M., and Dunican, L. K., Improved efficiency M13 cloning using
electroporation, Nuc. Acids Res., 17, 8006 (1989).
13. Holo, H. and Nes, I.F., Transformation of Lactococcus by electroporation, in
Methods in Molecular Biology, 47, Nickoloff, J.A. (ed.), Humana Press, Totowa, NJ, 195 (1995).
14. Howard, P.K., Ahern, K.G., and Firtel, R.A., Establishment of a transient
expression system for Dictyostelium discoideum, Nuc. Acids Res., 16, 2613 (1988).
15. Jacobs, M., Wnendt, S., and Stahl, U., High-efficiency electro-transformation of
Escherichia coli with DNA from ligation mixtures, Nuc. Acids Res., 18, 1653 (1990).
16. Knecht, D. and Pang, K.M., Electroporation of Dictyostelium discoideum, in
Methods in Molecular Biology, 47, Nickoloff, J.A., ed., Humana Press, Totowa, NJ, 321 (1995).
17. Lee, J.C., Electrotransformation of Staphylococci, in Methods in Molecular
Biology, 47, Nickoloff, J.A., ed., Humana Press, Totowa, NJ, 209 (1995). 18. Leonardo, E. D., and Sedivy, J. M., A new vector for cloning large eukaryotic
DNA segments in Escherichia coli, Bio/Technol., 8, 841 (1990).
19. Lin, J.-J., Electrotransformation of Agrobacterium, in Methods in Molecular
Biology, 47, Nickoloff, J.A., ed., Humana Press, Totowa, NJ, 171 (1995).
20. Miller, E.M. and Nickoloff, J.A., Escherichia coli electrotransformation, in
Methods in Molecular Biology, 47, Nickoloff, J.A., ed., Humana Press, Totowa, NJ, 105 (1995).
21. Nickoloff, J.A. (ed.) Methods in Molecular Biology, 47, Humana Press, Totowa,
NJ, (1995).
22. Park, S.F. and Stewart, G.S.A.B., High efficiency transformation of Listeria
monocytogenes by
23. electroporation of penicillin-treated cells, Gene, 94, 129 (1990).
24. Prentice, H.L., High efficiency transformation of Schizosaccharomyces pombe by
electroporation, Nuc. Acids Res., 20, 621 (1991).
25. Shigekawa, K. and Dower, W.J., Electroporation of eukaryotes and prokaryotes:
A general approach to the introduction of macromolecules into cells, Biotechniques, 6, 742 (1988).
26. Siguret, V., Ribba, A.-S., Cherel, G., Meyer, D., and Pieru, G., Effect of plasmid
size on transformation efficiency by electrtoporation of Escherichia coli DH5α, Biotechniques, 16, 422 (1994).
27. Simon, D. and Ferretti, J.J., Electrotransformation of Streptococcus pyogenes
with plasmid and linear DNA, FEMS Micobiol. Lett., 82, 219 (1991). 28. Somkuti, G.A. and Steinberg, Genetic transformation of Streptococcus
thermophilus by electroporation, Biochimie, 70, 579 (1988).
29. Summers, D. K., and Withers, H. L., Electrotransfer: direct transfer of bacterial
plasmid DNA by electroporation, Nuc. Acids Res., 18, 2192 (1990).
30. Taketo, A., DNA transfection of Escherichia coli by electroporation, Biochim.
Biophys. Acta, 949, 318 (1988).
31. Taketo, A., RNA transfection of Escherichia coli by electroporation, Biochim.
Biophys. Acta, 1007, 127 (1989).
32. Trevors, J.T., Chassy, B.M., Dower, W.J., and Blaschek, H.P.,
Electrotransformation of bacteria by plasmid DNA, in Guide to Electroporation and Electrofusion, Chang, D. C., Chassy, B. M., Saunders, J. A., and Sowers, A. E. (eds.), Academic Press Inc., 265, San Diego (1992).
33. Willson, T. A., and Gough, N. M., High voltage E. coli electro-transformation with
DNA following ligation, Nuc. Acids Res., 16, 11820 (1988).
34. Zabarovsky, E. R., and Winberg, G., High efficiency electroporation of ligated
DNA into bacteria, Nuc. Acids Res., 18, 5912 (1990).
Troubleshooting Guide for the Micropulser：略 产品信息
系统特点：
? 处理样品的速度更快——简单的一键“Pulse”操作、快速的充电时间。 ? 快速的程序选择——内置的、优化的程序适合细菌和酵母的通用研究。 ? 电弧淬灭系统（ARQ，Arc quenching system）——减少电弧效应，保护有价
值样本的损失。
? 更宽的参数手动优化空间——手动程序允许在200-3000V范围内设置电压，精
度10v；脉冲时间可以在1.0-4.0ms范围内调整，精度0.1ms。 ? 3000V容量——可以提高大体积的转化效率。 ? 紧凑型、节省空间的设计。 ? 可听见和可视化的脉冲指示。
? 可显示时间常数和实际释放电压——可监测重复性。
为什么要用电转化？
电转化是目前可用的效率最高的转化方法。它比化学转化方法的转化效率高几个数量级，
并且比其他方法重复性更好。MicroPulser被设计为E.coli、fungi和其他微生物的电转化提供最佳的电条件，可以获得最高的转化效率。