![]() (1994) Sepharose spin-column chromatography: a fast, nontoxic replacement for phenol:chloroform extraction/ethanol precipitation. Some plasmids may bear genes that code for. Importantly, transformants harboring correctly assembled constructs turn blue after incubation on media containing IPTG and X-Gal, while transformants harboring constructs with assembly. Klenow Fragment Klenow Fragment is the large fragment of DNA polymerase I. (1992) Converting restriction sites by filling in 5′ extensions. Briefly, the DNA assembly fragments comprise a cassette of the lac operon that is cloned into a destination vector containing an antibiotic resistance marker. (1991) Subcloning with new ampicillin and kanamycin resistant analogs of pUC19. (1984) Different restriction enzyme-generated sticky DNA ends can be Joined in vitro. (1984) Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli. (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA Proc. (1977) Construction and characterization of new cloning vehicles. ![]() Biotechniques 6, 929–932.īolivar, F., Rodriguez, R. (1988) Lid lysates an economical and rapid method for plasmid analysis. (1975) Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.ĭugaiczyk, A., Boyer, H. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Yanisch-Perron, C., Vieira, J., and Messing, J. Run the products on a gel, purify the desired fragments, and proceed with the common or compatible end restriction enzyme digestion of the destination vector. This process is experimental and the keywords may be updated as the learning algorithm improves. Simply digest the parent vector with your chosen restriction enzyme and use T4 DNA Polymerase or the DNA Polymerase I Klenow Fragment to make the end blunt. These keywords were added by machine and not by the authors. We describe here a variety of general subcloning strategies and protocols discussions of more specialized strategies may be found elsewhere (e.g., ref. ![]() Phage vectors, such as those based on M13 ( 1), do not use antibiotic markers instead transformants produce virus particles that kill or slow the growth of infected cells, and appear as plaques on lawns of uninfected bacteria. coli host cells), and one or more restriction sites into which foreign DNA may be inserted. Typically, cloning vectors have three essential elements, an antibiotic resistance marker, an origin of replication (to allow selection of transformed E. They are commonly used to construct expression systems, and to transfer fragments into specialized vectors for the preparation of hybridization probes and single-stranded DNA sequencing templates. Subcloning procedures are used to transfer DNA fragments from one vector context (plasmid, cosmid, or phage) to another. ![]()
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