DNA Learning Centers

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DNA from the Beginning
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Genetics Education Center
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DNA Learning Center
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Molecular Biology Laboratory Manual

Beginning Molecular Biology Laboratory Manual

CHAPTER 1: General Laboratory Methods

CHAPTER 2: Instructions for Notebook Keeping

CHAPTER 3: Vector NTI User's Guide

CHAPTER 4: Molecular Biology Methods

Preparation of genomic DNA from bacteria

PCR amplification of DNA

Restriction enzyme digestion of DNA

Phenol/chloroform extraction of DNA

Ethanol precipitation of DNA

Agarose gel electrophoresis

Transformation of E. coli by electroporation

Preparative DNA Fragment Isolation from an Agarose Gel

Ligations of plasmid DNA to insert DNA

Transfection of mammalian cells using Lipofectamine

Southern blotting

Western Blot analysis of His-tagged proteinsl

Cycle Sequencing Protocols For ABI 3100

Colony PCR

One Step Gene Assembly (Gene Synthesis)

CHAPTER 5: Tissue Culture Methods

Types of cells grown in culture

Work area and equipment

Preservation and storage

Maintenance

Safety considerations

Tissue culture procedures

Determining cell counts

Common Stock Solutions

CGH Protocols

Metaphase chromosome preparation
DNA preparation by cryostom tissue dissection
DNA labeling by nick translation
Hybridization
DNA detection
Image capture
Image analysis
DAPI chromosome identification

Production Sequencing Protocols

Production Sequencing Protocols used at the Stanford Genome Technology Center

Cosmid DNA Preparation

Lambda DNA Preparation

Pulsed-field Gel Purification of E. coli Restriction Fragments

Sequencing Library Construction

DNA Electroporation

Probe Construction

Hybridization

Template Preparation

Brew Recipe

ABI Catalyst 800

ABI 373 Sequencers

Sequence Assembly

Dye Terminator Sequencing

Roe Lab. Molecular Biology 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