• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        重组DNA的分离、克隆与测序实手册

        丁香园论坛

        1932
        PROTOCOLS FOR RECOMBINANT DNA ISOLATION, CLONING, and SEQUENCING
        edited by

        Bruce A. Roe
        Judy S. Crabtree
            and Akbar S. Khan
            Department of Chemistry and Biochemistry
        The University of Oklahoma
        Norman, Oklahoma 73019

        e-mail: broe@ou.edu
        phone: 405 325-4912
        fax: 405 325-7762
        August 5, 1995

        A printed version of this protocol book can be obtained from:

        "DNA Isolation and Sequencing" (Essential Techniques Series)
        by Bruce A. Roe, Judy S. Crabtree and Akbar S. Khan
        Published by John Wiley & Sons, List Price $49.95
        ISBN 0-471-97324-0
        QP625.N89R64 1996
        John Wiley & Sons

        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.

        Table of Contents
        I. General methods
        A. Phenol extraction of DNA samples
        B. Concentration of DNA by ethanol precipitation
        C. Restriction digestion
        D. Agarose gel electrophoresis
        E. Elution of DNA fragments from agarose
        F. Kinase end-labeling of DNA
        G. Bacterial cell maintenance
        H. Fragment purification on Sephacryl S-500 spin columns
        II. Random subclone generation
        A. Sonication
        B. Nebulization
        C. Random fragment end-repair, size selection, and phosphorylation
        D. DNA ligation
        E. Competent cell preparation
        F. Bacterial cell transformation
        G. Microcentrifuge tube transformation
        III. Methods for DNA isolation
        A. Large scale double-stranded DNA isolation
        B. Midiprep double-stranded DNA isolation
        C. Miniprep double-stranded DNA isolation
        D. Large scale M13RF isolation
        E. Single-stranded M13 DNA isolation using phenol
        F. Biomek-automated modified-Eperon isolation procedure for single-stranded M13DNA
        G. 96 well double-stranded template isolation
        H. Genomic DNA isolation from blood
        IV. Methods for DNA sequencing
        A. Bst-catalyzed radiolabeled DNA sequencing
        B. Radiolabeled sequencing gel preparation, loading, and electrophoresis
        C. Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye primers
        D. Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye terminator reactions
        1. Terminator Reaction Clean-Up via Centri-Sep Columns
        2. Terminator Reaction Clean-Up via Sephadex G-50 Filled Microtiter Format Filter Plates
        E. Sequenase[TM] catalyzed sequencing with dye-labeled terminators
        F. Fluorescent-labeled sequencing gel preparation, pre-electrophoresis, sample loading, electrophoresis, data collection, and analysis on the ABI 373A DNA sequencer
        G. Double-stranded sequencing of cDNA clones containing long poly(A) tails using anchored poly(dT) primers
        H. cDNA sequencing based on PCR and random shotgun cloning
        V. Additional methods
        A. Polymerase Chain Reaction (PCR)
        B. Purification of PCR fragments for cloning
        C. Preparation of SmaI-linearized, dephosphorylated double-stranded M13 replicative form cloning vector
        D. Synthesis and purification of oligonucleotides
        E. Rapid hybridization of complementary M13 inserts
        APPENDIX
        Solutions
        Primers
        Taq Cycle Sequencing Reagent Preparation
        Oligonucleotide universal primers used for DNA sequencing
        Listing of M13 (pUC) cloning sites
        Commonly used restriction enzymes and assay buffers
        Bacterial Transformation and Transfection
        Units and formulas
        DNA mobility in gels
        Codon chart and amino acid symbols
        Biomek configuration for single stranded DNA isolation
        Consensus sequences in nucleic acids
        References
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序