实验动物总则

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 ...

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 ...

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 ...

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 ...

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 ...

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 ( ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

Electroporation, originally developed as a method to introduce DNA into eukaryotic cells (1), has subsequently been extensively used for bacterial transformation (2,3). This procedure is an effective method for the transfer of DNA to a wide range of Gram-negative bacteria, such as Escher ...

Transformation is defined as the transfer of genetic information into a recipient bacterium using naked DNA, without any requirement for contact with a donor bacterium. The ability to transform or accept exogenous DNA is generally referred to as competence, although the term has been so wid ...

Bacterial conjugation is defined as contact-dependent transmission of genetic information from a donor bacterium to a recipient cell (1). Transfer of DNA by conjugation is often termed lateral or horizontal gene transfer, as opposed to vertical transfer by which genetic information is ...

Cosmids are cloning vectors that were developed to enable large fragments of DNA to be cloned and maintained (1–3). Cosmid vectors allow the cloning of fragments up to 45 kilobases (kb) and are commonly used in genomic library construction. The distinguishing feature of a cosmid is the presence of b ...

Purification of plasmid DNA from Escherichia coli using alkaline lysis (1,2) is based on the differential denaturation of chromosomal and plasmid DNA in order to separate the two. Bacteria are lysed with a solution containing sodium dodecyl sulfate (SDS) and sodium hydroxide. During this s ...