重组DNA的分离、克隆与测序实验手册借鉴.pdfVIP

PROTOCOLS FOR RECOMBINANT DNA ISOLATION, CLONING, and SEQUENCING 重组 DNA的分离、克隆与测序实验手册 Bruce A. Roe ， Judy S. Crabtree ， Akbar S. Khan Department of Chemistry and Biochemistry ，The University of Oklahoma Norman, Oklahoma 73019 Introduction This manual is a compilation of many of the everyday methods used in the average molecular biology laboratory, with emphasis on the techniques for large scale DNA sequencing protocols and DNA sequencing automation techniques. The manual has been written in a protocol format, with little theoretical discussion. For theory and additional information, users of this manual are referred back to the original literature, or to other textual manuals such as those published by Maniatis (1) et al. and Glover (2). The following persons are acknowledged for contributing methods and suggestions during the assembly of this manual: Stephanie Chissoe, Sandy Clifton, Dennis Burian, Rick Wilson, Din-Pow Ma, James Wong, Leslie Johnston-Dow, Elaine Mardis, Zhili Wang, Kala Iyer, Steve Toth, Goughay Zhang, Hua Qin Pan and other members of the Roe laboratory, both past and present. 1. Sambrook, J., Fritsch, E.F., and Maniatis, T., in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989). 2. Glover, D.M. DNA Cloning Volume I: A Practical Approach. IRL Press, Oxford, 1985. I. General methods A. Phenol extraction of DNA samples Phenol extraction is a common technique used to purify a DNA sample (1). Typically, an equal volume of TE-saturated phenol is added to an aqueous DNAsample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a newtube, avoiding the phenol interface and then is subjected to