文档介绍：PROTOCOLS FOR BINANT 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 pilation 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, ., and Maniatis, T., in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989).
2. Glover, . DNA Cloning Volume I: A Practical Approach. IRL Press, Oxford, 1985.
I. General methods
A. Phenol extraction of DNA samples
Phenol extraction is mon technique used to purify a DNA sample (1). Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample 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 new tube, avoiding the phenol interface and then is subjected to two ether extractions to remove residual phenol. An equal volume of water-saturated ether is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer