Beginning Molecular Biology Laboratory Manual
Preparation of genomic DNA from bacteria
Restriction enzyme digestion of DNA
Phenol/chloroform extraction of DNA
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
Western Blot analysis of His-tagged proteinsl
Cycle Sequencing Protocols For ABI 3100
One Step Gene Assembly (Gene Synthesis)
Production Sequencing Protocols used at the
Pulsed-field Gel Purification of E. coli Restriction Fragments
Table of Contents
A. Phenol extraction of DNA samples
B. Concentration of DNA by ethanol precipitation
D. Agarose gel electrophoresis
E. Elution of DNA fragments from agarose
H. Fragment purification on Sephacryl S-500 spin columns
II. Random subclone generation
C. Random fragment end-repair, size selection, and phosphorylation
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
H. cDNA sequencing based on PCR and random shotgun cloning
A. Polymerase Chain Reaction (PCR)
B. Purification of PCR fragments for cloning
D. Synthesis and purification of oligonucleotides
E. Rapid hybridization of complementary M13 inserts
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
Codon chart and amino acid symbols
Biomek configuration for single stranded DNA isolation
PCR screening of transformants (Crawford Lab.)
Bacterial Transformation and Transfection (Roe Lab.)
Preparation of Competent Cells
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 (
Bacterial Transformation (
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
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
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 ,
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.
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.
Some Interesting Uses of DNA Forensic Identification
Identifying September 11th Victims Identifying the victims of the September 11, 2001, World Trade Center attack presented a unique forensic challenge because the number and identity of the victims were unknown and many victims were represented only by bone and tissue fragments. At the time of the attack, no systems were in place for rapidly identifying victims in disasters with more than 500 fatalities. The National Institutes of Justice assembled a panel of experts from the National Institutes of Health and other institutions to develop processes to identify victims using DNA collected at the site. Panel members produced forms and kits needed to enable the medical examiner’s office to collect reference DNA from victims’ previously stored medical specimens. These specimens were collected and entered into a database. The medical examiner's office also received about 20,000 pieces of human remains from the World Trade Center site, and a database of the victims’ DNA profiles was created. New information technology infrastructure was developed for data transfer between the state police and medical examiner’s office and to interconnect the databases and analytical tools used by panel members. In 2005 the search was declared at an end because many of the unidentified remains were too small or too damaged to be identified by the DNA extraction methods available at that time. Remains of only 1585, of the 2792 people known to have died had been identified. In 2007, the medical examiner's office reopened the search after the Bode Technology Group developed a new methodology of DNA extraction that required much less sample material than previously necessary. The victim DNA database and the new methods have allowed more victims to be identified, and further identifications will be possible as forensic DNA technology improves.
The DNA Shoah ProjectThe DNA Shoah Project is a genetic database of people who lost family during the Holocaust. The database will serve to reunite families separated during wartime and aid in identifying victims who remain buried anonymously throughout Europe.
Disappeared Children in ArgentinaNumerous people (known as "the Disappeared") were kidnapped and murdered in Argentina in the 1970s. Many were pregnant. Their children were taken at birth and, along with other kidnapped children, were raised by their kidnappers. The grandparents of these children have been looking for them for many years. Read an article about a DNA researcher who has been helping them.
Tomb of the Unknowns
Son of Louis XVI and Marie AntionettePARIS, Apr 19, 2000 (Reuters) -- Scientists cracked one of the great mysteries of European history by using DNA tests to prove that the son of executed French King Louis XVI and Marie-Antoinette died in prison as a child. Royalists have argued for 205 years over whether Louis-Charles de France perished in 1795 in a grim Paris prison or managed to escape the clutches of the French Revolution. In December 1999, the presumed heart of the child king was removed from its resting place to enable scientists to compare its DNA makeup with samples from living and dead members of the royal family -- including a lock of his mother Marie-Antoinette's hair.
The Murdered Nicholas Romanov, the Last Czar of Russia, and His Family
Peruvian Ice MaidenThe Ice Maiden was a 12-to-14-year old girl sacrificed by Inca priests 500 years ago to satisfy the mountain gods of the Inca people. She was discovered in 1995 by climbers on Mt. Ampato in the Peruvian Andes. She is perhaps the best preserved mummy found in the Andes because she was in a frozen state. Analysis of the Ice Maiden's DNA offers a wonderful opportunity for understanding her genetic origin. If we could extract mitochondrial DNA from the Ice Maiden's tissue and successfully amplify and sequence it, then we could begin to trace her maternal line of descent and possibly locate past and current relatives.
African Lemba Tribesmen In southern Africa, a people known as the Lemba heed the call of the shofar. They have believed for generations that they are Jews, direct descendants of the biblical patriarchs Abraham, Isaac, and Jacob. However unlikely the Lemba's claims may seem, modern science is finding ways to test them. The ever-growing understanding of human genetics is revealing connections between peoples that have never been seen before.
Super Bowl XXXIV Footballs and 2000 Summer Olympic SouvenirsThe NFL used DNA technology to tag all the Super Bowl XXXIV balls, ensuring their authenticity for years to come and helping to combat the growing epidemic of sports memorabilia fraud. The footballs were marked with an invisible, yet permanent, strand of synthetic DNA. The DNA strand is unique and is verifiable any time in the future using a specially calibrated laser.
A section of human genetic code taken from several unnamed Australian athletes was added to ink used to mark all official goods — everything from caps to socks — from the 2000 Summer Olympic Games. The technology is used as a way to mark artwork or one-of-a-kind sports souvenirs.
Migration PatternsEvolutionarily stable mitochondrial DNA and Y chromosomes have allowed bioanthropologists to begin to trace human migration patterns around the world and identify family lineage
See Genetic Anthropology, Ancestry, and Ancient Human Migrations
Wine Heritage Using DNA fingerprinting techniques akin to those used to solve crimes and settle paternity suits, scientists at the University of California, Davis, have discovered that 18 of the world's most renowned grapevine varieties, or cultivars are close relatives. These include varieties long grown in northeastern France such as Chardonnay, the "king of whites," and reds such as Pinot and Gamay noir, are close relatives.
DNA Banks for Endangered Animal Species
Poached Animals
Declining Grizzly Bear Population
Snowball the Cat A woman was murdered in Prince Edward Island, Canada. Her estranged husband was implicated because a snowy white cat hair was found in a jacket near the scene of the crime, and DNA fragments from the hair matched DNA fragments from Snowball, the cat belonging to the husband's parents. See M. Menotti-Raymond et al., "Pet cat hair implicates murder suspect," Nature, 386, 774, 1997. Also see Holmes, Judy, Feline Forensics, Syracuse University Magazine, Summer 2001.
Angiosperm Witness for the Prosecution The first case in which a murderer was convicted on plant DNA evidence was described in the PBS TV series, "Scientific American Frontiers." A young woman was murdered in Phoenix, Arizona, and a pager found at the scene of the crime led the police to a prime suspect. He admitted picking up the victim but claimed she had robbed him of his wallet and pager. The forensic squad examined the suspect's pickup truck and collected pods later identified as the fruits of the palo verde tree (Cercidium spp.). One detective went back to the murder scene and found several Palo Verde trees, one of which showed damage that could have been caused by a vehicle. The detective's superior officer innocently suggested the possibility of linking the fruits and the tree by using DNA comparison, not realizing that this had never been done before. Several researchers were contacted before a geneticist at the University of Arizona in Tucson agreed to take on the case. Of course, it was crucial to establish evidence that would stand up in court on whether individual plants (especially Palo Verde trees) have unique patterns of DNA. A preliminary study on samples from different trees at the murder scene and elsewhere quickly established that each Palo Verde tree is unique in its DNA pattern. It was then a simple matter to link the pods from the suspect's truck to the damaged tree at the murder scene and obtain a conviction. [WNED-TV (PBS - Buffalo, N.Y.)]
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