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Studies on transformation of Escherichia coli with plasmids

High efficiency transformation of Escherichia coli with plasmids

We have isolated and sequenced a full-length cDNA clone encoding rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, E.C.1.2.1.12). The entire mRNA is 1269 nucleotides long exclusive of poly(A) and contains respectively 71 and 196 bases of 5' and 3' non-coding regions. Primer extension as well as S1 nuclease protection experiments clearly established that a single (or at least a highly prominent) GAPDH mRNA species is expressed in all rat tissues examined. This sequence allowed the determination of the hitherto unknown primary structure of rat GAPDH which is 333 aminoacids long. Comparison between GAPDH sequences from rat, man and chicken revealed a high degree of sequence conservation at both nucleotide and protein levels.

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Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family

Publisher Summary Oligonucleotide-directed in vitro mutagenesis provides a means to alter a defined site within a region of cloned DNA. This powerful technique has many far-reaching applications, from the definition of functional DNA sequences to the construction of new proteins. The method stemmed from the combination of a number of recent discoveries and observations about nucleic acids but the basic principle involves the enzymic extension by E. coli DNA polymerase of an oligonucleotide primer hybridized to a single-stranded circular template. More recently, phage M13 and M13 derived vectors have been used in a number of oligonucleotide mutagenesis experiments. Vectors derived from single-stranded phage, such as M13 and fd, are more convenient for oligonucleotide-directed mutagenesis than double-stranded vectors, because isolation of pure single-stranded circular template DNA in these systems is quite simple. This chapter describes a mutagenesis procedure that is simple, versatile, and efficient. The chapter also explains the targets and vehicles utilized in a number of recent mutagenesis experiments.

Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.

https://www.cell.com/cell/pdf/0092-8674(83)90037-5.pdf

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Papers

Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells.