PROTOCOLS FOR RECOMBINANT DNA ISOLATION, CLONING, and SEQUENCING

"DNA Isolation and Sequencing" (Essential Techniques Series)
by Bruce A. Roe, Judy S. Crabtree and Akbar S. Khan
Published by John Wiley & Sons, 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).

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

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