Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells.
TL;DR: Escherichia coli cells are 4--6 times more transformable and 20--30 times more competent after 24 h incubation in cold calcium chloride than immediately after calcium chloride treatment.
About: This article is published in Gene.The article was published on 1979-01-01. It has received 1354 citations till now. The article focuses on the topics: Calcium & Incubation.
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TL;DR: Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmid molecules.
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TL;DR: The conditions for preparing competent Escherichia coli cells are re-evaluated and a simple and efficient method (SEM) for plasmid transfection is established and these competent cells are particularly useful for construction of high-complexity cDNA libraries with a minimum expenditure of mRNA.
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TL;DR: This sequence allowed the determination of the hitherto unknown primary structure of rat GAPDH which is 333 aminoacids long and revealed a high degree of sequence conservation at both nucleotide and protein levels.
Abstract: 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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TL;DR: Oligonucleotide-directed in vitro mutagenesis provides a means to alter a defined site within a region of cloned DNA, from the definition of functional DNA sequences to the construction of new proteins.
Abstract: 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.
1,240 citations
TL;DR: The screened cDNA library for gene sequences that are regulated by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells indicates that these sequences correspond to "early genes" which are not induced as a consequence of cell growth, but rather are directly regulated by PDGF.
863 citations
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TL;DR: In vitro recombination techniques were used to construct a new cloning vehicle, pBR322, which is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc).
5,235 citations
TL;DR: Covalently-closed, catenated, and open (nicked) circular forms of R-factor DNA are all effective in transformation, but denaturation and sonication abolish the transforming ability of R.factor DNA in this system.
Abstract: Transformation of E. coli cells treated with CaCl2 to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before antibiotic challenge. Transformed bacteria acquire a closed circular, transferable DNA species having the resistance, fertility, and sedimentation characteristics of the parent R factor. Covalently-closed, catenated, and open (nicked) circular forms of R-factor DNA are all effective in transformation, but denaturation and sonication abolish the transforming ability of R-factor DNA in this system.
2,907 citations
TL;DR: Escherichia coli cells of strain K12 and C can be made competent to take up temperate phage DNA without the use of “helper phage”, and is effective for both linear and circular DNA molecules.
2,580 citations
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