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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

DNA (Gene) Transformation Protocols

Electrotransformation (Crawford Lab.)

PCR screening of transformants (Crawford Lab.)

Bacterial Transformation and Transfection (Roe Lab.)

Preparation of Competent Cells

Competent cell preparation

Method: Transformation of Plasmids/Cosmids into E. coli (Helen Donis-Keller Lab.)

Ligations in Low Melting Temperature Agarose (Helen Donis-Keller Lab)

Transformation of E. coli by electroporation (UMBC)

Electrocompetent E. coli preparation (Hahn Lab.)

Rubidium Chloride Method, NEB (BioLabs)

CaCl2 TRANSFORMATION OF E. coli (Hancock Lab.)

DNA Electroporation (Stanford DNA Center)

Bacterial Transformation (University of Arizona)

Transformation of E. coli with pGAL™ (blue colony)

Transformation of Bacteria by Plasmid DNA (Laboratoryexperimets.com)

Transformation of E. coli by Electroporation (Stanford)

Protocol - Chemical Transformation protocol (Baker Lab)

Transformation of Competent Cells (Laurie Lab)

Transformation Protocol for Arabidopsis - OpenWetWare

PCR Mutagenesis/Co-Transformation Protocol

Chen Lab Protocol--transformation

Chen Lab Protocol--transformation

High Efficiency Transformationmethod Page (TRAFO Page)

Yeast Transformation Protocol (by Sudhir Nayak)

Transformation Protocol (Laurie Lab.)

High Electrotransformation Efficiencies Obtained With DNA From Ligation Mixtures

http://www.biocompare.com/technicalarticle/1137/High-Electrotransformation-Efficiencies-Obtained-With-DNA-From-Ligation-Mixtures-from-Bio-Rad.html

Selected Articles on Mutagenesis

MW Unger , SY Liu , and DE Rancourt

Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination
Nucl. Acids Res. 27: 1480-1484.

Paul Gaytán , Jorge Yáñez , Filiberto Sánchez , and Xavier Soberón

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols
Nucl. Acids Res. 29: e9.

RD Kirsch , and E Joly

An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes
Nucl. Acids Res. 26: 1848-1850.

Qing Lin , Sarah L. Donahue , Tracy Moore-Jarrett , Shang Cao , Anna B. Osipovich , and H. Earl Ruley

Mutagenesis of diploid mammalian genes by gene entrapment
Nucleic Acids Research Advance Access published on November 6, 2006, DOI 10.1093/nar/gkl728.
Nucl. Acids Res. 34: e139.

Igor Shevelev , Giuseppina Blanca , Giuseppe Villani , Kristijan Ramadan , Silvio Spadari , Ulrich Hübscher , and Giovanni Maga

Mutagenesis of human DNA polymerase : essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities
Nucl. Acids Res. 31: 6916-6925.

Jaesung Lee , and David L. Herrin

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth
Nucl. Acids Res. 31: 4361-4372.

Michael O’Connor , Wyan-Ching Mimi Lee , Anuj Mankad , Catherine L. Squires , and Albert E. Dahlberg

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable
Nucl. Acids Res. 29: 710-715.

Eun Ju Lee , Joel Glasgow , Sew-Fen Leu , Ali O. Belduz , and James G. Harman

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 the cyclic nucleotide binding pocket
Nucl. Acids Res. 22: 2894-2901.

M.J. Pillaire , A. Margot , G. Villani , A. Sarasin , M. Defias , and A. Gentil

Mutagenesis in monkey cells of a vector containing a single d(GPG) cis-diamminedichloroplatinum(ll) adduct placed on codon 13 of the human H-ras proto-oncogen
Nucl. Acids Res. 22: 2519-2524.

Pamela Anderson , Joseph Monforte , Richard Tritz , Steven Nesbitt , John Hearst , and Arnold Hamper

Mutagenesis of the hairpin ribozyme
Nucl. Acids Res. 22: 1096-1100.

Huey-Nan Wu , Jun-YU Lee , Huey-Wen Huang , Yi-shuian Huang , and Tung-Guang Hsueh

Mutagenesis analysis of a hepatitis delta virus genomic ribozyme
Nucl. Acids Res. 21: 4193-4199.

Ali O. Belduz , Eun Ju Lee , and James G. Harman

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli targeting positions 72 and 82 of the cyclic nucleotide binding pocket
Nucl. Acids Res. 21: 1827-1835.

Huey-Nan Wu , and Zhi-Shun Huang

Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA
Nucl. Acids Res. 20: 5937-5941.

V. Pletsa , A. Gentil , A. Margot , J. Armier , S.A. Kyrtopoulos , and A. Sarasin

Mutagenesis by 06 meG residues within codon 12 of the human Ha-ras proto-oncogene in monkey cells
Nucl. Acids Res. 20: 4897-4901.

Akira Sekiguchi , Yasuo Komatsu , Makoto Koizumi , and Eiko Ohtsuka

Mutagenesis and self-ligation of the self-cleavage domain of the satellite RNA minus strand of tobacco rinspot virus and its binding to polyamines
Nucl. Acids Res. 19: 6833-6838.

Aidan J. Doherty , Andrew F. Worall , and Bernard A. Connolly

Mutagenesis of the DNA binding residues in bovine pancreatic DNase1: an investigation into the mechanism of sequence discrimination by a sequence selective nuclease
Nucl. Acids Res. 19: 6129-6132.

Christian Baron , Johann Heider , and August Böck

Mutagenesis of selC, the gene for the selenocysteine-insertlng tRNA-species in E.coli: effects on in vivo function
Nucl. Acids Res. 18: 6761-6766.

Candice C. Sheldon , and Robert H. Symons

Mutagenesis analysis of a self-cleaving RNA
Nucl. Acids Res. 17: 5679-5685.

Jacques Piette , Howard B. Gamper , Albert Van de Vorst , and John E. Hearst

Mutagenesis induced by site specifically placed 4'-hydroxymethyl-4,5',8-trimethylpsoralen adducts
Nucl. Acids Res. 16: 9961-9977.

G. Chua , L. Taricani , W. Stangle , and P. G. Young

Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe
Nucl. Acids Res. 28: e53.

MJ Singer , MA Podyminogin , MA Metcalf , MW Reed , DA Brown , HB Gamper , RB Meyer , and RM Wydro

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides
Nucl. Acids Res. 27: e38.

A Melnikov , and PJ Youngman

Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus
Nucl. Acids Res. 27: 1056-1062.

RM Tucker , and DT Burke

Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol
Nucl. Acids Res. 24: 3467-3468.

David P. Siderovski , Toshifumi Matsuyama , Elena Frigerio , Stephen Chui , Xia Min , Heather Erfle , Martin Sumner-Smith , Richard W. Bamett , and Tak W. Mak

Random mutagenesis of the human immunodeficiency virus type-1 frans-activator of transcription (HIV-1 Tat)
Nucl. Acids Res. 20: 5311-5320.

Xiubei Liao , David Selinger , Steven Althoff , Anne Chiang , Diana Hamilton , Min Ma , and Jo Ann Wise

Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites
Nucl. Acids Res. 20: 1607-1615.

Yuhong Zhou , Xiaoping Zhang , and Richard H. Ebright

Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase
Nucl. Acids Res. 19: 6052.

Bruno A. Gaëta , Stephen J. Sharp , and Thomas S. Stewart

Saturation mutagenesis of the Drosophila tRNAArg gene B-Box intragenic promoter element: requirements for transcription activation and stable complex formation
Nucl. Acids Res. 18: 1541-1548.

Laurent Dulau , Alain Cheyrou , and Michel Aigle

Directed mutagenesis using PCR
Nucl. Acids Res. 17: 2873.

John M. Hinz , Robert S. Tebbs , Paul F. Wilson , Peter B. Nham , Edmund P. Salazar , Hatsumi Nagasawa , Salustra S. Urbin , Joel S. Bedford , and Larry H. Thompson

Repression of mutagenesis by Rad51D-mediated homologous recombination
Nucl. Acids Res. 34: 1358-1368.

Tuck Seng Wong , Kang Lan Tee , Berhard Hauer , and Ulrich Schwaneberg

Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution
Nucl. Acids Res. 32: e26.

Gordana Maravic , Janusz M. Bujnicki , Marcin Feder , Sándor Pongor , and Mirna Flögel

Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein–RNA interactions
Nucl. Acids Res. 31: 4941-4949.

Fumi Nagatsugi , Shigeki Sasaki , Paul S. Miller , and Michael M. Seidman

Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog
Nucl. Acids Res. 31: e31.

Assen Marintchev , Michael R. Gryk , and Gregory P. Mullen

Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group 1, with DNA polymerase ß
Nucl. Acids Res. 31: 580-588.

P. Soultanas , and D. B. Wigley

Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus
Nucl. Acids Res. 30: 4051-4060.

MS Dillingham , P Soultanas , and DB Wigley

Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation
Nucl. Acids Res. 27: 3310-3317.

P Neuner , R Cortese , and P Monaci

Codon-based mutagenesis using dimer-phosphoramidites
Nucl. Acids Res. 26: 1223-1227.

B Hallet , DJ Sherratt , and F Hayes

Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein
Nucl. Acids Res. 25: 1866-1867.

W Wu , C Palaniappan , RA Bambara , and PJ Fay

Differences in mutagenesis during minus strand, plus strand and strand transfer (recombination) synthesis of the HIV-1 gene in vitro
Nucl. Acids Res. 24: 1710-1718.

Gil Barzilay , Lisa J. Walker , Craig N. Robson , and Ian D. Hickson

Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity
Nucl. Acids Res. 23: 1544-1550.

Chung-Nam Chung , Yasushi Hamaguchi , Tasuku Honjo , and Masashi Kawaichi

Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-Jx
Nucl. Acids Res. 22: 2938-2944.