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  

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