Roe Lab. Protocols

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