By Jac A. Nickoloff
This really good choice of well-tested protocols for the electroporation of proteins and DNA into insect, fish, and mammalian cells additionally contains confirmed animal phone electrofusion innovations for experiences of somatic mobile genetics and improvement, and for producing monoclonal antibodies. it's exotic via its assurance of significant version cellphone varieties from many organisms and tissue varieties and its distinct directions for the expansion and education of particular cells to accomplish optimal transfection. Its huge reference lists, citations of different move tools, suggestion on pitfalls to prevent, and outlines of anticipated effects make sure easily reproducible effects even for novices.
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Extra info for Animal Cell Electroporation and Electrofusion Protocols
Chang, D. , Gao, P. , and Maxwell, B. L (1991) High efficiency gene transfection by electroporation using a radio-frequency electric field. Biochim. Btophys. Actu 1992,153-160. 20 Tekle, E , Austumlan, R. , and Chock, P. B (1991) Electroporatron by using bipolar oscillating electric field. an improved method for DNA transfectlon of NIH 3T3 cells. Proc. Natl. Acad. Sci USA 88,4230-4234 CHAPTER3 Instrumentation Gunter A. Hofmann 1. Introduction The techniques of electroporation and electrofusion require that cells be subjected to brief pulses of electric fields of the appropriate amplitude, duration, and wave form.
L. and Evans, G. A (1989) Optimization of electroporatlon transfection of mammalian cells. Anal. Biochem 180,269-275. for 40 Hui 8. , Klenchin, V. , and Chizmadzhev, Y. A. (1992) Electroporation and electrophoretic DNA transfer mto cells The effect of DNA interaction with electropores Bzophys. J. 63,1320-1327 9. Xie, T. , and Tsong, T Y. (1990) Study of mechamsms of electrrc fieldinduced DNA transfectron I DNA entry by surface binding and diffusion through membrane pores. Biophys. J. 58, 13-19.
Metaphase Cell Chromosome Aberration Assay 1. At various times after electroporation, add colcemid to exponentially growing cells at a final concentration of 2 x 10e7M(see Note 7). 2. Collect metaphase cells after l-3 h by gently shaking the flask. 3. 075M) KC1 prewarmed to 37°C. 4. Incubate the cells at 37°C for 5-15 min, then recentrifuge, and resuspend them m methanol for 2 min. 5. Centrifuge the cells, and resuspend them m methanol-acetic acid (3:l) for 5 min. 6. Centrifuge the cells, and resuspend them in a small volume of methanolacetic acid.
Animal Cell Electroporation and Electrofusion Protocols by Jac A. Nickoloff