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Using Desktop Cloning Software to Plan, Track, and Evaluate Cloning Projects

Manipulation and analysis of DNA sequences is often a complex task involving many steps, each of which must be carefully planned and executed. To facilitate this process, the number of steps should be minimized and each step analyzed to ensure that it has been completed successfully. Often, this a ...

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Cloning in Plasmid Vectors

A fundamental step in molecular biology is the cloning of a DNA fragment insert into a plasmid vector. This allows the cloned fragment to be replicated upon transformation of the recombinant molecule into a bacterial cell (see Chapters 4 and 5) so that the DNA of interest can be investigated further. C ...

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Extraction of DNA from Agarose Gels

A common step in cloning experiments is the purification of DNA fragments prior to ligation. Often, both the insert and vector DNA fragments will be derived from restriction endonuclease digests and, thus, will be mixed with enzymes, salts, and possibly other DNA fragments that may inhibit the en ...

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Cloning PCR Products with T-Vectors

Since it was described in 1988 (1), the polymerase chain reaction (PCR) has been a valuable tool for molecular biologists. PCR allows researchers to produce a large quantity of a desired DNA fragment while requiring only a small amount of template. Prior to PCR, isolation of DNA fragments was typical ...

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Construction of Genomic Libraries in -Vectors

Lambda (λ) bacteriophages are viruses that specifically infect bacteria. The genome of λ-phage is a double-stranded DNA molecule approx 50 kb in length (1). In bacterial cells, λ-phage employs one of two pathways of replication: lytic or lysogenic. Commonly, λ-phage vectors replicate via the l ...

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Rapid Screening of Recombinant Plasmids

Construction of recombinant plasmid DNA is one of the cornerstones of molecular biology. The ability to clone DNA in a plasmid vector opens doors to downstream applications such as amplification of DNA, expression of desired genes, and construction of DNA libraries. Recombinant plasmids ...

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Restriction Analysis of Recombinant Plasmids

A key step in the construction of recombinant plasmids is the verification of the successful cloning of insert DNA into the vector. A number of commonly used plasmids facilitate phenotypic selection and/or screening methods for rapid identification of insert-containing clones. Addi ...

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The Function and Organization of Plasmids

In 1952, Joshua Lederberg coined the term plasmid to describe any bacterial genetic element that exists in an extrachromosomal state for at least part of its replication cycle (1). As this description included bacterial viruses, the definition of what constitutes a plasmid was subsequent ...

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Screening Recombinant DNA Libraries

A recombinant DNA library typically represents part or all of an organism’s genomic DNA or mRNA (represented as cDNA) cloned into vectors and stored as a collection of thousands of transformants. The construction of a complete library is only half the task; researchers then need to be able to identi ...

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Sequencing Using Fluorescent-Labeled Nucleotides

The most widespread method used for DNA sequencing today is the Sanger dideoxy method that was first described in 1977 (1). This method takes advantage of the requirement for a free 3′ hydroxyl group to form the necessary phosphodiester bridge between two nucleotides during DNA polymerizati ...

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Site-Directed Mutagenesis Using the Megaprimer Method

Site-directed mutagenesis (SDM) is used to introduce a defined mutation into target DNA of known sequence to study, for example, gene expression or protein structure–function relationship. A number of polymerase chain reaction (PCR)-based mutagenesis methods have been developed ( ...

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Site-Directed Mutagenesis by Inverse PCR

Site-directed mutagenesis has revolutionized the study of protein structure and function by enabling the controlled and systematic production of mutant proteins. Early methods of site-directed mutagenesis involved the use of a mutated oligonucleotide primer to prime synthe ...

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Transposon and Transposome Mutagenesis of Plasmids, Cosmids, and BACs

Transposons are mobile genetic elements with the capacity to “jump” to new target DNA. Although first discovered in Zea mays by McClintock (1), they are present in DNA genomes of species from all kingdoms. Transposons fall into two major classes. Class I transposons are retroelements that trans ...

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Creating Nested DNA Deletions Using Exonuclease III

DNA fragments cloned into plasmids are frequently greater than 500 base pairs in length and thus may be too long to sequence from a single primer-binding site in the vector. An efficient way to sequence such large DNA inserts is to generate a nested set of deletions in the target DNA, effectively moving the ...

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Vectors for the Expression of Recombinant Proteins in E. coli

Escherichia coli is the most commonly used and best characterized organism for overexpressing foreign and nonforeign proteins. The use of E. coli confers several immediate advantages to the user: rapid and high-level expression as a result of the speed of cell growth to high density; low compl ...

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Expression of Recombinant Proteins From lac Promoters

The Gram-negative bacterium Escherichia coli enjoys widespread use in modern biology as both a model organism and a microbiological tool. One of the keys to its popularity lies in the functionality of the lac operon. This regulated E. coli operon has been manipulated and utilized in diverse and cr ...

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Choosing a Cloning Vector

Since the construction of the first generation of general cloning vectors in the early 1970s, the number of plasmids created has increased to an almost countless number. Thus, a critical decision facing today’s investigator is that of which plasmid to use in a particular project? Despite the bew ...

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Plasmid-Based Reporter Genes: Assays for -Galactosidase and Alkaline Phosphatase Activities

Reporter genes encode easily measurable traits. Most commonly, they are used to investigate the expression of other genes for which functional assays are not available or for which measurement of expressed product is difficult. This is often done through constructing a genetic fusion in w ...

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Plasmid-Based Reporter Genes: Assays for Green Fluorescent Protein

Green fluorescent protein (GFP) of the jellyfish Aqueorea victoria is a 238-amino-acid, 28-kDa protein that absorbs light with an excitation maximum of 395 nm and fluoresces with an emission maximum of 509 nm (1). GFP owes its unique spectral properties to its chromophore (2) that consists of a Ser6 ...

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Escherichia coli Host Strains

To successfully perform molecular genetic techniques it is essential to have a full understanding of the properties of the various Escherichia coli host strains commonly used for the propagation and manipulation of recombinant DNA. E. coli is an enteric rod-shaped Gram-negative bacte ...

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