Protocols For Recombinant DNA Isolation, Cloning, and Sequencing
http://www.genome.ou.edu/protocol_book/protocol_index.html
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
Bruce A. Roe, Department of Chemistry and Biochemistry, The University
of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu